WO2020027309A1 - Intranasal vaccine that induces cellular immunity - Google Patents

Intranasal vaccine that induces cellular immunity Download PDF

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Publication number
WO2020027309A1
WO2020027309A1 PCT/JP2019/030399 JP2019030399W WO2020027309A1 WO 2020027309 A1 WO2020027309 A1 WO 2020027309A1 JP 2019030399 W JP2019030399 W JP 2019030399W WO 2020027309 A1 WO2020027309 A1 WO 2020027309A1
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antigen
vaccine
gene product
cyclic
cells
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PCT/JP2019/030399
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French (fr)
Japanese (ja)
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義和 幸
理佳 中橋
宏 清野
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国立大学法人東京大学
株式会社ＨａｎａＶａｘ
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Application filed by 国立大学法人東京大学, 株式会社ＨａｎａＶａｘ filed Critical 国立大学法人東京大学
Priority to CA3107263A priority Critical patent/CA3107263A1/en
Priority to JP2020534761A priority patent/JP7445897B2/en
Priority to EP19844843.3A priority patent/EP3831403A4/en
Priority to CN201980051194.9A priority patent/CN112566656A/en
Priority to AU2019313996A priority patent/AU2019313996A1/en
Priority to US17/265,267 priority patent/US11564993B2/en
Priority to KR1020217004785A priority patent/KR20210040387A/en
Publication of WO2020027309A1 publication Critical patent/WO2020027309A1/en

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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
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    • A61P31/12—Antivirals
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a nasal vaccine that induces cell-mediated immunity.
  • Humoral immunity is an immune system centered on antibodies, complement, etc., which are mainly present in the blood.
  • antigen presenting cells such as dendritic cells take it in, fragment it, and present it on the cell surface via MHC class II molecules.
  • the Th2 cells stimulated by the antigen-presenting cells recognize the antigen fragment presented on the B cells via the T cell antigen receptor (TCR), and release Th2 cytokines and the like.
  • B cells produce antibodies under the action of released Th2 cytokines.
  • cell-mediated immunity is an immune system that eliminates foreign substances in a living body by macrophages, cytotoxic T lymphocytes (CTLs), natural killer cells, and the like.
  • CTLs cytotoxic T lymphocytes
  • Th1 cells When Th1 cells are activated by antigen fragments presented on antigen presenting cells via MHC class II molecules, they release IFN- ⁇ to activate macrophages.
  • ADCC Antibody-Dependent-Cellular-Cytotoxicity
  • activated Th1 cells release IL-2 and activate CTLs that recognize antigen fragments presented with MHC class I molecules.
  • cCHP cholesteryl group-bearing pullulan
  • Patent Literature 1 Non-Patent Literature 1
  • cCHP nanogel functions as an artificial chaperone, prevents aggregation and denaturation of the antigen, and helps refolding after antigen release.
  • This nanogel has the property of efficiently adhering to the negatively charged mucosal surface, and induces an immune response by continuously releasing the antigen and delivering the antigen to antigen presenting cells (Non-Patent Document 2, Non-Patent Document 2). Reference 3 and Patent Document 2).
  • Nanogel vaccines suitable for nasal administration are very good both in terms of safety and induction of humoral immunity. However, it has not been confirmed so far to induce cell-mediated immunity.
  • an object of the present invention is to provide a nanogel nasal vaccine that induces cell-mediated immunity.
  • the present inventors have developed a vaccine in which a STING ligand is encapsulated in a nanogel as an adjuvant, in addition to a vaccine antigen, and when administered intranasally to mice, to induce the antigen-specific Th1 cells, in order to solve the above problems. Succeeded.
  • the present invention includes the following (1) to (11).
  • a vaccine preparation comprising a complex of a nanogel, a vaccine antigen and an adjuvant.
  • the cyclic dinucleotide is any one of cGAMP, cyclic-di AMP, cyclic-di GMP, cyclic-di CMP, cyclic-di UMP or cyclic-di IMP.
  • the antigen derived from Mycobacterium tuberculosis is at least the whole of Ag85B gene product, Rv2608 gene product, Rv3619 gene product, Rv3620 gene product, Rv1813 gene product, MTB32A gene product, MTB39A gene product and / or MVA85A gene product.
  • HPV human papillomavirus
  • the HPV-derived antigen contains at least the whole or a part of the E6 gene product and / or the E7 gene product.
  • the vaccine preparation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from respiratory syncytial virus (RSV).
  • RSV-derived antigen contains at least the whole or a part of the SH peptide.
  • ⁇ Cellular immunity can be induced by administering the nanogel vaccine according to the present invention.
  • cGMP, cGAMP and cAMP stand for cyclic-di-GMP, cyclic-GMP-AMP and cyclic-di-AMP, respectively.
  • - No vaccine ⁇ ⁇ ⁇ ⁇ cCHP : cationic cholesteryl-group bearing pullulan Detection result of Th1 cell response induced by Nagel Mycobacterium tuberculosis nasal vaccine. Detection result of Th17 cell response induced by Nagel Mycobacterium tuberculosis nasal vaccine. Examination of the protective immunity effect by the Nagel Mycobacterium tuberculosis nasal vaccine.
  • (A) shows the survival rate
  • (B) shows the number of M. tuberculosis detected from lung and spleen.
  • “Control” is a group of unimmunized mice
  • BCG is a group vaccinated with BCG
  • nanogel is a group vaccinated with cCHP-Ag85B + cyclic-di-GMP.
  • Detection result of Th1 cell response induced by Mycobacterium tuberculosis nasal vaccine chimeric antigen.
  • Detection results of CTL cell response induced by Nanogel HPV nasal vaccine. Detection results of Th1 cell response induced by Nanogel HPV nasal vaccine.
  • a first embodiment of the present invention is a vaccine preparation (hereinafter also referred to as “the vaccine preparation of the present invention”) comprising a complex of a nanogel, a vaccine antigen and an adjuvant.
  • the nanogel is a polymer gel nanoparticle in which hydrophobic cholesterol is added as a side chain to a hydrophilic polysaccharide (for example, pullulan).
  • the nanogel can be produced based on a known method, for example, the method described in International Publication WO00 / 12564.
  • a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms and a diisocyanate represented by OCN-R1 NCO (where R1 is a hydrocarbon group having 1 to 50 carbon atoms)
  • the compound is reacted to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms has reacted.
  • the obtained isocyanate group-containing hydrophobic compound is reacted with a polysaccharide to produce a hydrophobic group-containing polysaccharide having a hydrocarbon group or a steryl group having 12 to 50 carbon atoms.
  • a highly pure hydrophobic group-containing polysaccharide can be produced.
  • the polysaccharide pullulan, amylopectin, amylose, dextran, hydroxyethyldextran, mannan, levan, inulin, chitin, chitosan, xyloglucan or water-soluble cellulose can be used, and pullulan is particularly preferable.
  • cCHP cationic cholesteryl-substituted pullulan
  • cCHP has a structure in which pullulan having a molecular weight of 30,000 to 200,000, for example, molecular weight of 100,000 is substituted with 1 to 10, preferably 1 to several cholesterol per 100 monosaccharides.
  • the cholesterol substitution amount may be appropriately changed depending on the size and hydrophobicity of the antigen.
  • an alkyl group (10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms) may be added.
  • the nanogel used in the present invention has a particle size of 10 to 40 nm, preferably 20 to 30 nm. Nanogels are already widely commercially available, and these commercially available products may be used.
  • the nanogel used in the embodiment of the present invention is a nanogel into which a functional group having a positive charge, for example, an amino group has been introduced so that the vaccine can enter the surface of the negatively charged nasal mucosa.
  • a method for introducing an amino group into the nanogel include a method using cholesterol pullulan (CHPNH 2 ) to which an amino group is added. Specifically, CHP dried under reduced pressure is dissolved in dimethyl sulfoxide (DMSO), and 1-1'carbonyldiimidazole is added thereto under a nitrogen stream, and the mixture is reacted for several hours at room temperature. Ethylenediamine is gradually added to the reaction solution and stirred for several hours to several tens of hours.
  • DMSO dimethyl sulfoxide
  • the obtained reaction solution is dialyzed against distilled water for several days.
  • the reaction solution after dialysis is freeze-dried to obtain a milky white solid.
  • the degree of substitution of ethylenediamine can be evaluated using elemental analysis, H-NMR and the like.
  • the vaccine antigen is not particularly limited, and can be arbitrarily selected according to the use of the vaccine preparation.
  • the vaccine preparation according to the present invention can induce cell-mediated immunity efficiently, and is therefore very suitable for use in activating the cellular immune system in preventing or treating diseases and the like.
  • diseases are tuberculosis, for which there is no effective vaccine for adults, tuberculosis-free Haemophilus influenzae (NTHi), RSV (respiratory syncytial virus) infection, or HSV (herpes virus simple virus), for which there is no effective vaccine for adults.
  • Infectious diseases or HPV (human papilloma virus) infections for which induction of cell-mediated immunity is considered to be important for the treatment thereof, and cervical cancer caused by the infection.
  • Examples of the vaccine antigen for tuberculosis include, but are not limited to, Ag85B (Rv1886) gene product, ESAT6 (Rv3875) gene product, Rv2660 gene product, Rv2608 gene product, Rv3619 gene product, and Rv3620 derived from Mycobacterium tuberculosis.
  • Examples of vaccine antigens for non-capsulated Haemophilus influenzae include D15, P1, P2, P4, P5, P6, Hmw / hia, Hap, Protein E, ProteinF, ProteinD, Pil A, NucA, HtrA, OMP26, PCP , TbpB or LOS, or a part thereof, or a plurality of fusion proteins selected from these proteins.
  • the RSV vaccine antigen is not particularly limited.
  • RSV-derived F protein (fusion protein) or whole or a part of SH protein, or a plurality of fusion proteins selected from these proteins may be used. Good.
  • HSV vaccine antigens are not particularly limited, but include, for example, HSV-derived gD gene product, gB gene product, gC gene product, gE gene product, capsid protein UL19, Tegment protein UL47 or the whole or one of gG gene products. Or a plurality of fusion proteins selected from these proteins.
  • HPV vaccine antigen include, but are not limited to, HPV-derived E6 gene product, particularly a mutation or deletion product at the E6 binding site of tumor suppressor gene product P53, HPV-derived E7 gene product, particularly cancer suppression.
  • HPV6 E7 23-27 deletion
  • HPV11 E7 23-27 deletion
  • HPV16 E7 D21G, C24G, E26G mutation
  • HPV18 E7 24-27 deficiency
  • HPV31 E7 22-26 deficiency
  • HPV33 E7 22-26 deficiency
  • HPV45 E7 26-30 deficiency
  • HPV52 E7 22 -26 deficient
  • a part thereof or a plurality of fusion proteins selected from these proteins.
  • the adjuvant used in the embodiment of the present invention is synonymous with what is called an antigenic enhancer or an immunopotentiator, and is used in the art for the usual purpose of using these agents. .
  • the active ingredient of the adjuvant used in the embodiment of the present invention is not particularly limited.
  • STING ligands that activate STING eg, cGAMP, cyclic-di AMP, cyclic-di GMP, Cyclic dinucleotides such as cyclic-di CMP, cyclic-di UMP or cyclic-di IMP, xanthenone (Xanthenone) derivatives such as DMXAA (5,6-dimethylXAA (xanthenone-4- acetic acid), Vadimezan or ASA404), polyIC Alternatively, CpG-ODN and the like can be mentioned.
  • STING ligands that activate STING eg, cGAMP, cyclic-di AMP, cyclic-di GMP, Cyclic dinucleotides such as cyclic-di CMP, cyclic-di UMP or cyclic-di IMP, xanthenone (Xanthenone) derivatives such as DMXAA (5,6-dimethylXAA (xanthenone-4-
  • the adjuvant may further contain a pharmaceutically acceptable carrier and other components (for example, a stabilizer, a pH adjuster, a preservative, a preservative, a buffer and the like).
  • a pharmaceutically acceptable carrier and other components for example, a stabilizer, a pH adjuster, a preservative, a preservative, a buffer and the like.
  • Pharmaceutically acceptable carriers and other ingredients need to be substances that do not adversely affect the health of the animal being vaccinated.
  • the complex of the nanogel, the vaccine antigen and the adjuvant (or the active ingredient of the adjuvant, the same applies hereinafter) is prepared by allowing the nanogel, the vaccine antigen and the adjuvant to coexist and interact, and incorporating the antigen and the adjuvant into the nanogel.
  • the mixing ratio of the nanogel and the vaccine antigen, and the mixing ratio of the nanogel and the adjuvant are not particularly limited, and can be easily determined by those skilled in the art through preliminary experiments.
  • the vaccine antigen: nanogel ratio is, for example, about 0.1: 10, 1: 5, 1: 2 or 1: 1 in molar ratio.
  • the content of the adjuvant may be about 0.01% to 99.99% by weight based on 100% by weight of the vaccine, and may be, for example, about 0.01% to 10% by weight based on 1% of the antigen.
  • the formation of the complex of the nanogel, the vaccine antigen and the adjuvant is performed by mixing the nanogel, the vaccine antigen and the adjuvant, and allowing the mixture to stand at 4 to 50 ° C, for example, 40 ° C, for 30 minutes to 48 hours, for example, about 1 hour. can do.
  • the buffer used for forming the complex of the nanogel, the vaccine antigen and the adjuvant is not particularly limited, and for example, a Tris-HCl buffer and the like can be mentioned.
  • the vaccine preparation of the present invention may contain a pharmacologically acceptable additive as a composition (the vaccine composition of the present invention).
  • the vaccine preparation of the present invention is suitable for nasal administration, and is desirably in a form capable of nasal administration, and examples thereof include liquid preparations (nasal drops and injections).
  • the active ingredient is optionally adjusted with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate and sodium dihydrogen phosphate, sodium chloride and It is dissolved in distilled water for preparation together with an isotonic agent such as glucose, and then sterile-filtered and filled into ampoules, or mannitol, dextrin, cyclodextrin, gelatin, etc. are added and freeze-dried in vacuum, and the working-soluble preparation is prepared. It may be.
  • a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate and sodium dihydrogen phosphate, sodium chloride and It is dissolved in distilled water for preparation together with an isotonic agent such as glucose, and then sterile-filtered and filled into ampoules, or mannitol, dextrin, cyclodextrin, gelatin, etc. are
  • the liquid formulation may contain known pharmaceutically acceptable stabilizers, preservatives, antioxidants, and the like.
  • stabilizers include gelatin, dextran, sorbitol, and the like.
  • thimerosal and ⁇ -propiolactone for example, thimerosal and ⁇ -propiolactone, and as the antioxidant, for example, ⁇ -tocopherol and the like.
  • a second embodiment of the present invention is directed to a method for preventing and / or treating a disease, comprising intranasally administering to a patient a vaccine formulation (first embodiment) comprising a complex of a nanogel, a vaccine antigen and an adjuvant. is there.
  • the disease to be treated or prevented in the second embodiment depends on the vaccine antigen to be used and is not particularly limited.
  • infectious diseases caused by pathogens eg, tuberculosis, HSV and RSV, etc.
  • cancers eg, Cervical cancer
  • the vaccine formulation of the present invention may be administered via the nasal mucosa. Examples of the method include a method in which the drug is administered into the nasal cavity by spraying, applying, dripping, or the like on the nasal mucosa.
  • the dose of the mucosal vaccine preparation can be appropriately determined depending on the age, weight, and the like of the administration subject, and includes a pharmaceutically effective amount of the vaccine antigen.
  • a pharmaceutically effective amount refers to that amount of antigen required to induce an immune response to the vaccine antigen.
  • the vaccine antigen may be administered once to several times a day at a dose of several ⁇ g to several tens mg, and may be administered several times at intervals of one to several weeks, for example, about 1 to 5 times.
  • the extracted protein fraction is charged to a nickel affinity column (GE Healthcare Bio-Sciences), washed with an adsorption buffer until the OD 280 nm becomes 0.01 or less, 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole and 6 M urea.
  • the protein was eluted with a solution containing Next, the eluate was concentrated with Amicon, and subjected to gel filtration with a Sephacryl S-100 column (GE Healthcare Bio-Sciences) equilibrated with 6M-Urea PBS, and the Ag85B fraction was collected, and 4M-Urea PBS, 2M- Urea PBS, 1M-Urea PBS and PBS were dialyzed stepwise to prepare native Ag85B. 50 mg of Ag85B (SEQ ID NO: 2) was recovered in a 12 L culture of E. coli, and the purity was 95% by SDS-PAGE.
  • Nanogelation of antigen preparation of vaccine
  • the cCHP nanogel was prepared according to the method described previously (Non-Patent Document 2). After mixing the prepared cCHP nanogel and the purified Ag85B protein at a molecular ratio of 1: 1 and further adding three types of STING ligands (cyclic-di-GMP, cyclic-di-AMP and cGAMP) as adjuvants, Incubated for 1 hour in a 40 ° C. heat block.
  • STING ligands cyclic-di-GMP, cyclic-di-AMP and cGAMP
  • cCHP nanogel and chimeric purified protein (ESAT6-Rv2660c-Rv0288) (amino acid sequence: SEQ ID NO: 8, nucleic acid sequence: SEQ ID NO: 9) were mixed at a molecular ratio of 1: 1, and further, as a mucosal adjuvant, STING ligand (cyclic-cyclic) was used. After adding di-AMP), the mixture was incubated in a heat block at 40 ° C. for 1 hour.
  • CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells.
  • CD4-positive T cells and gamma-irradiated antigen-presenting cells were co-cultured under purified Ag85B antigen stimulation for 48-72 hours.
  • an anti-IFN ⁇ or anti-IL-17 antibody was previously adsorbed to the bottom of the culture well as a capture antibody.
  • a biotin-labeled anti-IFN ⁇ antibody or anti-IL-17 antibody was added, and reacted at room temperature for 2 hours. After that, the wells were washed and reacted with streptavidin HRP. After washing, HRP substrate 3-Amino-9-ethylcarbazole (AEC) was added to develop color, and antigen-specific Th1 cells or Th17 cells were detected as spots. . The number of spots was measured using an ELISPOT counter.
  • ESAT6-Rv2660c-Rv0288 chimeric antigen Two weeks after the last administration, antigen-specific Th1 cells (IFN ⁇ -producing cells) were counted by the ELISPOT method. The systemic immune response was evaluated in the spleen, and the mucosal immune response was evaluated using antigen-specific T cells generated in lung tissue. After the mouse was euthanized, the lung and spleen were removed and the cell suspension was prepared. From there, CD4 positive T cells were purified using magnetic beads. On the other hand, CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells.
  • CD4-positive T cells and gamma-irradiated antigen presenting cells were co-cultured for 48-72 hours under the stimulation of purified chimeric antigen or recombinant ESAT6 (Abcam).
  • ESAT6 purified chimeric antigen or recombinant ESAT6
  • anti-IFN ⁇ was used as a capture to detect production cells.
  • streptavidin HRP was reacted, and after washing, 3-Amino-9-ethylcarbazole (AEC), a substrate of HRP, was reacted to develop color, and antigen-specific Th1 was detected as a spot.
  • AEC 3-Amino-9-ethylcarbazole
  • mice were 7-week-old Balb / c females.
  • the BCG vaccine as a positive control was suspended in a PBS solution and administered subcutaneously once to mice at the time of the first immunization.
  • the mixed solution of cCHP-Ag85B + cyclic-di-GMP was nasally administered at a dose of 10 ⁇ g per animal once per week at an interval of 10 ⁇ g of Ag85B protein.
  • Unimmunized control mice received PBS intranasally three times every other week and once subcutaneously at the time of the first immunization.
  • HPV vaccine 2-1 Preparation of antigen protein
  • the three amino acids D21G, C24G and E26G mutation E7 (Van der Burg SH et.al. Vaccine 19: 3652-3660, 2001) of the tumor suppressor gene product of HPV16 virus (307 bp) (SEQ ID NO: 3) were artificially generated. It was synthesized and inserted into EcoRI-HinIII (Takara Bio Inc.) site of a pET-20b (+) vector (Novagen) having a gene of His-Tag sequence. The prepared expression vector was transformed into Rosetta2 (DE3) pLysS-E. Coli by a conventional method.
  • the obtained transformant was cultured in a medium containing 100 ⁇ g / mL ampicillin and 34 ⁇ g / mL chloramphenicol at 37 ° C. until the OD600nm reached 0.5-0.8. Thereafter, 1.0 mM isopropyl ⁇ -D-1-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added, and the cells were cultured for 4 hours. The cultured E. coli was collected by centrifugation (5,000 rpm, 15 minutes). The collected E.
  • coli was washed with a solution containing 10 mM imidazole and protease inhibitor (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole and 6 M urea.
  • the extracted protein fraction is charged to a nickel affinity column (GE Healthcare Bio-Sciences), washed with an adsorption buffer until the OD 280 nm becomes 0.01 or less, 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole and 6 M urea.
  • the protein was eluted with a solution containing Subsequently, the eluate was dialyzed against 6 M Urea-PBS (0.15 M NaCl), adsorbed on a DEAE-sepharose column (GE Healthcare Bio-Sciences KK) equilibrated with the same buffer, and then 0.5 M NaCl-PBS-6 M Urea Eluted with a solution containing The eluate was concentrated with Amicon, gel-filtered through a Sephacryl S-100 column (GE Healthcare Bio-Sciences) equilibrated with 6M-Urea PBS, and the mutant E7 fraction was collected, and 4M-Urea PBS, 2M -Urea PBS, 1M-Urea PBS and PBS were dialyzed stepwise to prepare native mutant E7 (SEQ ID NO: 4). 60 mg of mutant E7 was recovered in 12 L of E. coli culture, and the purity was 95% by SDS-PAGE.
  • Nanogelation of antigen preparation of vaccine
  • the cCHP nanogel was prepared according to the method described previously (Non-Patent Document 2).
  • the prepared cCHP nanogel and the purified mutant E7 protein are mixed at a molecular ratio of 1: 1.
  • only adjuvant cyclic-di-AMP and three kinds of STING ligands cyclic-di-GMP, cyclic-di-AMP, cGAMP
  • poly I C, CpG ODN K3 or D35, respectively, and then incubated for 1 hour in a heat block at 40 ° C.
  • Nasal Immunization to Mice A mixed solution of cCHP-mutant E7 and each mucosal adjuvant was intranasally administered to 7-week-old female Balb / c mice. The amount of the antigen to be administered was 10 ⁇ g in terms of the amount of mutant E7 protein per animal. Each mucosal adjuvant was administered at 5 ⁇ g or 10 ⁇ g per animal. Nasal immunization was performed three times at weekly intervals.
  • CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells.
  • Purified T cells and gamma-irradiated antigen presenting cells were co-cultured under purified mutant E7 antigen stimulation for 48-72 hours.
  • an anti-granzyme B antibody or an anti-IFN ⁇ antibody was previously adsorbed to the bottom of the culture well as a capture antibody.
  • a biotin-labeled anti-granzyme B antibody or anti-IFN ⁇ antibody was added, and the mixture was reacted at room temperature for 2 hours. Then, the wells were washed and reacted with streptavidin HRP. After washing, HRP substrate 3-Amino-9-ethylcarbazole (AEC) was added to develop color, and antigen-specific CTL cells or Th1 cells were detected as spots. . The number of spots was measured using an ELISPOT counter.
  • Purified T cells and gamma-irradiated antigen-presenting cells were co-cultured for 48-72 hours under the stimulation of purified mutant E7 antigen.
  • An anti-IFN ⁇ antibody or an anti-granzyme B antibody was laid on the bottom of the culture well as a capture to detect production cells. After removing the culture supernatant, the wells were washed and reacted with a biotin-labeled anti-IFN ⁇ antibody or anti-granzyme B antibody. Further, after washing, streptavidin HRP was reacted, and after washing, AEC which is a substrate of HRP was reacted to develop color, and antigen-specific Th1 or CTL was detected as a spot. The spot was measured by an ELISPOT counter.
  • RSV vaccine 3-1 Preparation of antigen protein
  • This plasmid was transformed into Rosetta2 (DE3) pLysS-E. Coli by a conventional method.
  • This Escherichia coli was cultured in a medium containing 100 ⁇ g / mL ampicillin and 34 ⁇ g / mL chloramphenicol at 37 ° C. until the OD600 nm reached 0.5-0.8. Thereafter, 1.0 mM isopropyl ⁇ -D-1-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added, and after culturing for 4 hours, Escherichia coli was recovered by centrifugation (5000 rpm, 15 minutes).
  • the bacteria were washed with a solution containing 20 mM imidazole and protease inhibitor (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20 mM Tris-HCl, 500 mM NaCl, and 10 mM imidazole.
  • An adsorption buffer containing 20 mM Tris-HCl, 500 mM NaCl, and 10 mM imidazole.
  • a saturated ammonium sulfate solution was added to the extract to 80% saturation, and ammonium sulfate precipitation was performed.
  • the precipitate was collected by centrifugation, and dialyzed using the same buffer solution as used for extraction as the external solution.
  • the dialyzed solution is charged to a nickel affinity column (GE Healthcare Bio-Sciences), washed with an adsorption buffer until the OD 280 nm becomes 0.01 or less, and the solution containing 20 mM Tris-HCl, 500 mM NaCl, and 500 mM imidazole is used. Eluted.
  • the eluate was concentrated with Amicon, gel-filtered through a Sephadex G-100 column (GE Healthcare Bio-Sciences) equilibrated with PBS, and the PspA-SH3 fraction was collected, concentrated and purified. 70 mg of PspA-SH3 was recovered by culturing 20 L of E. coli, and the purity was 95% by SDS-PAGE.
  • Nanogelation of antigen preparation of vaccine
  • cCHP nanogel and PspA-SH3 purified protein SEQ ID NO: 6
  • SEQ ID NO: 6 Nanogelation of antigen (preparation of vaccine)
  • cCHP nanogel and PspA-SH3 purified protein SEQ ID NO: 6
  • SEQ ID NO: 6 cyclic-di-AMP was added as a mucosal adjuvant, followed by incubation in a heat block at 40 ° C. for 1 hour.
  • Nasal Immunization to Mice A mixed solution of cCHP-PspA-SH3 + cyclic-di-AMP was intranasally administered to a 7-week-old female Balb / c mouse. The amount of antigen to be administered was 10 ⁇ g of PspA-SH3 protein per animal. Moreover, 10 ug of cyclic-di-AMP was administered. Nasal immunization was performed three times at weekly intervals, once every four weeks, and once every four weeks for a total of five times.
  • Each sample was placed in 1% BSA-containing PBS-T with 2 of 8-fold diluted those plates end well, to prepare a dilution series for 2-fold serial dilutions to the other end, was incubated for 2 hours at room temperature .
  • the blank was PBS-T containing 1% BSA. After the incubation, the plate was washed four times with 300 ⁇ L of PBS-T using a plate washer.
  • TMB Substrate and TMB Solution were added at 100 ⁇ l / well, and after 30 minutes of color development reaction, 50 ⁇ l of 2N H 2 SO 4 (nacalai tesque, 32520-55) was added. The reaction was stopped. The value of OD450 was measured with a plate reader, and the value of log2titer was calculated. The cut-off value was the average value of the blank well + 0.1.
  • Mycobacterium tuberculosis vaccine (1) Ag85B antigen 10 ⁇ g of cyclic-di-GMP compared to the case where a mixture of 10 ⁇ g of CpGK3, which is known to exhibit adjuvant activity, and 1 ⁇ g of cGAMP, which is a kind of STING ligand, is added. In a similar degree, induction of antigen-specific Th1 cell-mediated immunity was observed (FIG. 1). In comparison with the STING ligand alone (cAMP, GMP, and cGAMP), cyclic-di-AMP (cAMP) appeared to be relatively effective.
  • Cyclic-di-GMP was used as a STING ligand. Mice to which a vaccine antigen containing no Cyclic-di-GMP was administered hardly induced antigen-specific Th1 cells and Th17 cells (FIGS. 2 and 3 "cCHP-Ag85B"). T cells were hardly induced even when the antigen-presenting cells were not stimulated with the antigen.
  • the BCG vaccine was examined as a positive control for the effect on the survival rate and growth of M. tuberculosis when the intranasal nanogel vaccine of the present invention using the STING ligand as an adjuvant was administered to mice.
  • the survival rate was calculated from the fact that several deaths were observed from the 12th week after infection to 56% of non-immunized mice (negative control) and 67% of BCG vaccine group (positive control).
  • the nanogel group cCHP-Ag85B + cyclic-di-GMP administration
  • the number of M. tuberculosis bacteria in the spleen was significantly significantly suppressed in the BCG and nanogel vaccine groups as compared with the non-immunized mice, and a similar tendency was observed in the lungs (FIG. 4B).
  • HPV vaccine (1) In the case of using cyclic-di-AMP as an adjuvant
  • the nanogel nasal vaccine of the present invention was prepared using HPV mutant E7 protein as an antigen, and the effect of inducing the vaccine on T cells and the like was examined.
  • Nasal administration of cCHP-mutant E7 + cyclic-di-AMP was found to induce antigen-specific CTL in the spleen and cervix (FIG. 6).
  • intranasal administration of cCHP-mutant E7 + cyclic-di-AMP induced antigen-specific Th1 cells in the spleen and cervix (FIG. 7).
  • IgG1 In the IgG subclass, in both the anti-SH peptide and the anti-PspA antibody, IgG1 was predominant without cyclic-di-AMP adjuvant, and IgG1 and IgG2b were induced with adjuvant (FIG. 10).
  • T cells characteristic of cellular immunity such as Th1 cells and CLT, were induced. Furthermore, it was also found that it induces mucosal immunity not only in systemic immunity but also in genital mucosal tissues in addition to upper respiratory and lower respiratory tract mucosal tissues.
  • the nanogel nasal vaccine of the present invention can induce cell-mediated immunity and is expected to be used in medical fields such as immunocell therapy.

Abstract

The present invention provides a nanogel intranasal vaccine that induces cellular immunity. Specifically, the present invention relates to a vaccine formulation that comprises a complex of a nanogel, a vaccine antigen, and an adjuvant. The vaccine formulation can efficiently induce cellular immunity and also can induce a systemic and mucosal immune response.

Description

細胞性免疫を誘導する経鼻ワクチンNasal vaccine that induces cell-mediated immunity
　本発明は、細胞性免疫を誘導する経鼻ワクチンに関する。 The present invention relates to a nasal vaccine that induces cell-mediated immunity.
　獲得免疫は、液性免疫および細胞性免疫という異なる２つの機構によって担われている。
　液性免疫は、主として血中に存在する抗体および補体等を中心とする免疫システムである。生体内に外来抗原が侵入すると、樹状細胞などの抗原提示細胞がこれを取り込んで断片化した後、MHCクラスII分子を介してその細胞表面上に提示する。その後、抗原提示細胞からの刺激を受けたTh2細胞が、T細胞抗原受容体（TCR）を介してB細胞上に提示された抗原断片を認識し、Th2サイトカインの放出等を行う。B細胞は、放出されたTh2サイトカインの作用を受けて抗体を産生する。
　他方、細胞性免疫は、マクロファージ、細胞傷害性T細胞（cytotoxic T Lymphocytes：CTL）およびナチュラルキラー細胞などにより生体内の異物を排除する免疫システムである。MHCクラスII分子を介して抗原提示細胞上に提示された抗原断片によりTh1細胞が活性化されると、IFN-γを放出して、マクロファージを活性化する。また中和抗体ではなく、細胞表面に結合する抗体を誘導し抗体のFcレセプターを介して、マクロファージやNK細胞を活性化させ標的細胞を攻撃し破壊するADCC（Antibody-Dependent-Cellular-Cytotoxicity）の誘導も考えられる。加えて、活性化されたTh1細胞はIL-2を放出し、MHCクラスI分子と共に提示された抗原断片を認識したCTLを活性化する。活性化されたマクロファージおよびCTLは、ウイルス等に感染した細胞やがん細胞などを攻撃し排除する。細胞性免疫は、感染細胞やがん細胞などの排除も可能であることから、細胞内に寄生することが可能な結核菌の排除や、がん免疫療法への応用が期待されている。 Acquired immunity is played by two different mechanisms: humoral immunity and cellular immunity.
Humoral immunity is an immune system centered on antibodies, complement, etc., which are mainly present in the blood. When a foreign antigen invades the living body, antigen presenting cells such as dendritic cells take it in, fragment it, and present it on the cell surface via MHC class II molecules. Thereafter, the Th2 cells stimulated by the antigen-presenting cells recognize the antigen fragment presented on the B cells via the T cell antigen receptor (TCR), and release Th2 cytokines and the like. B cells produce antibodies under the action of released Th2 cytokines.
On the other hand, cell-mediated immunity is an immune system that eliminates foreign substances in a living body by macrophages, cytotoxic T lymphocytes (CTLs), natural killer cells, and the like. When Th1 cells are activated by antigen fragments presented on antigen presenting cells via MHC class II molecules, they release IFN-γ to activate macrophages. In addition, ADCC (Antibody-Dependent-Cellular-Cytotoxicity), which induces antibodies that bind to the cell surface instead of neutralizing antibodies and activates macrophages and NK cells to attack and destroy target cells via the antibody Fc receptor Induction is also conceivable. In addition, activated Th1 cells release IL-2 and activate CTLs that recognize antigen fragments presented with MHC class I molecules. The activated macrophages and CTL attack and eliminate cells infected with a virus or the like or cancer cells. Since cell-mediated immunity can eliminate infected cells and cancer cells, it is expected to eliminate tuberculosis bacteria that can be parasitic in cells and to be applied to cancer immunotherapy.
　これまでに発明者らは、コレステロールが付加されたカチオン性プルランによって構成される自己凝集性ナノサイズヒドロゲル（cCHP；cationic type of cholesteryl group-bearing pullulan）を利用して、効果的なワクチンの送達システムを開発した（特許文献１、非特許文献１）。cCHPナノゲルは、そのナノマトリックス内部にタンパク質抗原を内包すると、人工的なシャペロンとして機能し、抗原の凝集および変性を防ぎ、抗原放出後のリフォールディングを助ける。このナノゲルは、効率的に負電荷の粘膜表面に付着する性質を持ち、持続的に抗原を放出して抗原提示細胞まで抗原を送達することで免疫応答を誘導する（非特許文献２、非特許文献３および特許文献２）。また、マウスにおいて、 [111In]-標識 BoHc/A（ボツリヌスA型毒素の重鎖C末端領域無毒領域） や肺炎球菌表面抗原PspAを担持するcCHPナノゲルを経鼻的に投与しても、嗅球や脳などの中枢神経系に蓄積することはなく（非特許文献２）、その安全性も確認されている（非特許文献４）。 Heretofore, the present inventors have proposed an effective vaccine delivery system using a cationic type of cholesteryl group-bearing pullulan (cCHP) composed of cationic pullulan to which cholesterol has been added. (Patent Literature 1, Non-Patent Literature 1). When a protein antigen is encapsulated inside its nanomatrix, cCHP nanogel functions as an artificial chaperone, prevents aggregation and denaturation of the antigen, and helps refolding after antigen release. This nanogel has the property of efficiently adhering to the negatively charged mucosal surface, and induces an immune response by continuously releasing the antigen and delivering the antigen to antigen presenting cells (Non-Patent Document 2, Non-Patent Document 2). Reference 3 and Patent Document 2). Moreover, in mice, nasal administration of [ 111 In] -labeled BoHc / A (non-toxic region of the heavy chain C-terminal region of botulinum type A toxin) or cCHP nanogel carrying pneumococcal surface antigen PspA It does not accumulate in the central nervous system such as the brain or the brain (Non-Patent Document 2), and its safety has been confirmed (Non-Patent Document 4).
　経鼻投与に適したナノゲルワクチン（ナノゲル経鼻ワクチン）は、安全性および液性免疫の誘導の両面において非常に良好である。
　しかしながら、これまでのところ細胞性免疫を誘導することは確認されてない。 Nanogel vaccines suitable for nasal administration (nasal gels) are very good both in terms of safety and induction of humoral immunity.
However, it has not been confirmed so far to induce cell-mediated immunity.
WO00/12564号WO00 / 12564 特許第5344558号Patent No. 5344558
　上記事情に鑑み、本発明は、細胞性免疫を誘導するナノゲル経鼻ワクチンの提供を目的とする。 In view of the above circumstances, an object of the present invention is to provide a nanogel nasal vaccine that induces cell-mediated immunity.
　本発明者らは、上記課題を解決するために、ワクチン抗原の他、アジュバントとしてSTINGリガンドをナノゲルに封入したワクチンを作成し、マウスに経鼻投与したところ、抗原特異的なTh1細胞を誘導することに成功した。 The present inventors have developed a vaccine in which a STING ligand is encapsulated in a nanogel as an adjuvant, in addition to a vaccine antigen, and when administered intranasally to mice, to induce the antigen-specific Th1 cells, in order to solve the above problems. Succeeded.
　すなわち、本発明は、以下の（１）～（１１）である。
（１）ナノゲル、ワクチン抗原およびアジュバントの複合体を含むワクチン製剤。
（２）前記アジュバントが１または複数のSTINGリガンドを含むことを特徴とする上記（１）に記載のワクチン製剤。
（３）前記STINGリガンドの少なくとも１つが、環状ジヌクレオチドであることを特徴とする上記（２）に記載のワクチン製剤。
（４）前記環状ジヌクレオチドが、cGAMP、cyclic-di AMP、cyclic-di GMP、cyclic-di CMP、cyclic-di UMPまたはcyclic-di IMPのいずれかであることを特徴とする上記（３）に記載のワクチン製剤。
（５）前記ワクチン抗原が結核菌由来の抗原であることを特徴とする上記（１）ないし（４）のいずれかに記載のワクチン製剤。
（６）前記結核菌由来の抗原が、少なくともAg85B遺伝子産物、Rv2608遺伝子産物、Rv3619遺伝子産物、Rv3620遺伝子産物、Rv1813遺伝子産物、MTB32A遺伝子産物、MTB39A遺伝子産物および／またはMVA85A遺伝子産物の全体もしくはその一部を含むことを特徴とする上記（５）に記載のワクチン製剤。
（７）前記結核菌由来の抗原が、Rv3875遺伝子産物、Rv0266遺伝子産物およびRv0288遺伝子産物からなるキメラタンパク質であることを特徴とする上記（５）に記載のワクチン製剤。
（８）前記ワクチン抗原がHPV（human papillomavirus）由来の抗原であることを特徴とする上記（１）ないし（４）のいずれかに記載のワクチン製剤。
（９）前記HPV由来の抗原が少なくともE6遺伝子産物および／またはE7遺伝子産物の全体もしくはその一部を含むことを特徴とする上記（８）に記載のワクチン製剤。
（１０）前記ワクチン抗原がRSV（respiratory syncytial virus）由来の抗原であることを特徴とする上記（１）ないし（４）のいずれかに記載のワクチン製剤。
（１１）前記RSV由来の抗原が少なくともSHペプチドの全体もしくはその一部を含むことを特徴とする上記（１０）に記載のワクチン製剤。 That is, the present invention includes the following (1) to (11).
(1) A vaccine preparation comprising a complex of a nanogel, a vaccine antigen and an adjuvant.
(2) The vaccine preparation according to the above (1), wherein the adjuvant contains one or more STING ligands.
(3) The vaccine preparation according to the above (2), wherein at least one of the STING ligands is a cyclic dinucleotide.
(4) The above-mentioned (3), wherein the cyclic dinucleotide is any one of cGAMP, cyclic-di AMP, cyclic-di GMP, cyclic-di CMP, cyclic-di UMP or cyclic-di IMP. The vaccine preparation according to the above.
(5) The vaccine preparation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from Mycobacterium tuberculosis.
(6) The antigen derived from Mycobacterium tuberculosis is at least the whole of Ag85B gene product, Rv2608 gene product, Rv3619 gene product, Rv3620 gene product, Rv1813 gene product, MTB32A gene product, MTB39A gene product and / or MVA85A gene product. The vaccine preparation according to the above (5), comprising a part.
(7) The vaccine preparation according to the above (5), wherein the Mycobacterium tuberculosis-derived antigen is a chimeric protein comprising an Rv3875 gene product, an Rv0266 gene product, and an Rv0288 gene product.
(8) The vaccine preparation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from HPV (human papillomavirus).
(9) The vaccine preparation according to the above (8), wherein the HPV-derived antigen contains at least the whole or a part of the E6 gene product and / or the E7 gene product.
(10) The vaccine preparation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from respiratory syncytial virus (RSV).
(11) The vaccine preparation according to the above (10), wherein the RSV-derived antigen contains at least the whole or a part of the SH peptide.
　本発明にかかるナノゲルワクチンの投与により、細胞性免疫を誘導することができる。 細胞 Cellular immunity can be induced by administering the nanogel vaccine according to the present invention.
　本発明にかかるナノゲルワクチンの投与により、全身性免疫応答および粘膜免疫応答の両方を効率よく誘導することができる。 投 与 Both the systemic immune response and the mucosal immune response can be efficiently induced by administering the nanogel vaccine according to the present invention.
ナノゲル結核菌経鼻ワクチンとSTINGリガンドにより誘導されるTh1細胞応答の検出結果。cGMP、cGAMPおよびcAMPは、各々、cyclic-di-GMP、cyclic-GMP-AMPおよびcyclic-di-AMPを表す。－：ワクチン無し　cCHP：cationic cholesteryl-group bearing pullulanDetection result of Th1 cell response induced by M. tuberculosis nasal vaccine and STING ligand. cGMP, cGAMP and cAMP stand for cyclic-di-GMP, cyclic-GMP-AMP and cyclic-di-AMP, respectively. -: No vaccine ワ ク チ ン cCHP ： cationic cholesteryl-group bearing pullulan ナノゲル結核菌経鼻ワクチンにより誘導されるTh1細胞応答の検出結果。Detection result of Th1 cell response induced by Nagel Mycobacterium tuberculosis nasal vaccine. ナノゲル結核菌経鼻ワクチンにより誘導されるTh17細胞応答の検出結果。Detection result of Th17 cell response induced by Nagel Mycobacterium tuberculosis nasal vaccine. ナノゲル結核菌経鼻ワクチンによる防御免疫効果の検討。（A）は生存率を示し、（B）は肺および脾臓から検出された結核菌数を示す。「コントロール」は未免疫マウス群、「BCG」はBCGワクチン接種群、「ナノゲル」はcCHP-Ag85B+cyclic-di-GMP接種群である。Examination of the protective immunity effect by the Nagel Mycobacterium tuberculosis nasal vaccine. (A) shows the survival rate, and (B) shows the number of M. tuberculosis detected from lung and spleen. “Control” is a group of unimmunized mice, “BCG” is a group vaccinated with BCG, and “Nanogel” is a group vaccinated with cCHP-Ag85B + cyclic-di-GMP. ナノゲル結核菌経鼻ワクチン（キメラ抗原）により誘導されるTh1細胞応答の検出結果。Detection result of Th1 cell response induced by Mycobacterium tuberculosis nasal vaccine (chimeric antigen). ナノゲルHPV経鼻ワクチンにより誘導されるCTL細胞応答の検出結果。Detection results of CTL cell response induced by Nanogel HPV nasal vaccine. ナノゲルHPV経鼻ワクチンにより誘導されるTh1細胞応答の検出結果。Detection results of Th1 cell response induced by Nanogel HPV nasal vaccine. 3種のSTINGリガンドをアジュバントとして用いたナノゲルHPV経鼻ワクチンにより誘導されるCTL細胞応答（左）およびTh1細胞応答（右）の比較。Comparison of CTL cell response (left) and Th1 cell response (right) induced by a nanogel HPV nasal vaccine using three STING ligands as adjuvants. ナノゲルRSV経鼻ワクチンにより誘導される免疫応答の検出結果。Detection results of immune response induced by Nanogel RSV nasal vaccine. ナノゲルRSV経鼻ワクチンにより誘導されるIgGサブクラスの検出結果。Detection results of IgG subclass induced by Nanogel RSV nasal vaccine.
　本発明の第１の実施形態は、ナノゲル、ワクチン抗原およびアジュバントの複合体を含むワクチン製剤（以下「本発明のワクチン製剤」とも記載する）である。
　本発明において、ナノゲルとは、親水性の多糖（例えば、プルラン）に、側鎖として疎水性のコレステロールが付加された、高分子ゲルナノ粒子のことである。ナノゲルは公知の方法、例えば、国際公開第WO00/12564号公報に記載された方法などに基づいて製造することができる。
　具体的には、まず、炭素数12～50の水酸基含有炭化水素またはステロールと、OCN-R1 NCO（式中、R1は炭素数1～50の炭化水素基である）で表されるジイソシアナート化合物を反応させて、炭素数12～50の水酸基含有炭化水素またはステロールが1分子反応したイソシアナート基含有疎水性化合物を製造する。得られたイソシアナート基含有疎水性化合物と多糖類とを反応させ、炭素数12～50の炭化水素基またはステリル基を含有する疎水性基含有多糖類を製造する。次に、得られた生成物をケトン系の溶媒で精製することにより、純度の高い疎水性基含有多糖類を製造することができる。
　ここで、多糖類としては、プルラン、アミロペクチン、アミロース、デキストラン、ヒドロキシエチルデキストラン、マンナン、レバン、イヌリン、キチン、キトサン、キシログルカンまたは水溶性セルロース等が利用可能であり、特に、プルランが好ましい。 A first embodiment of the present invention is a vaccine preparation (hereinafter also referred to as “the vaccine preparation of the present invention”) comprising a complex of a nanogel, a vaccine antigen and an adjuvant.
In the present invention, the nanogel is a polymer gel nanoparticle in which hydrophobic cholesterol is added as a side chain to a hydrophilic polysaccharide (for example, pullulan). The nanogel can be produced based on a known method, for example, the method described in International Publication WO00 / 12564.
Specifically, first, a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms and a diisocyanate represented by OCN-R1 NCO (where R1 is a hydrocarbon group having 1 to 50 carbon atoms) The compound is reacted to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms has reacted. The obtained isocyanate group-containing hydrophobic compound is reacted with a polysaccharide to produce a hydrophobic group-containing polysaccharide having a hydrocarbon group or a steryl group having 12 to 50 carbon atoms. Next, by purifying the obtained product with a ketone-based solvent, a highly pure hydrophobic group-containing polysaccharide can be produced.
Here, as the polysaccharide, pullulan, amylopectin, amylose, dextran, hydroxyethyldextran, mannan, levan, inulin, chitin, chitosan, xyloglucan or water-soluble cellulose can be used, and pullulan is particularly preferable.
　本発明の第１の実施形態で使用されるナノゲルとしては、カチオン性コレステロール置換プルラン（cationic cholesteryl-group-bearing pullulan：cCHPと称する）およびその誘導体を挙げることができる。cCHPは、分子量3万から20万、例えば分子量100,000のプルランに100単糖あたりコレステロールが1～10個、好ましくは1～数個置換された構造を有する。なお、本発明で使用されるcCHPは、抗原のサイズや疎水性の度合いにより、コレステロール置換量を適宜変更してもよい。また、CHPの疎水性の度合いを変更するために、アルキル基（炭素数10～30、好ましくは、炭素数12～20程度）を付加させてもよい。本発明で使用されるナノゲルは、粒径10～40nm、好ましくは20～30nmである。ナノゲルは既に広く市販されており、これら市販品を使用してもよい。 {Examples of the nanogel used in the first embodiment of the present invention include cationic cholesteryl-substituted pullulan (hereinafter referred to as cCHP) and its derivative. cCHP has a structure in which pullulan having a molecular weight of 30,000 to 200,000, for example, molecular weight of 100,000 is substituted with 1 to 10, preferably 1 to several cholesterol per 100 monosaccharides. In the cCHP used in the present invention, the cholesterol substitution amount may be appropriately changed depending on the size and hydrophobicity of the antigen. In order to change the degree of hydrophobicity of CHP, an alkyl group (10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms) may be added. The nanogel used in the present invention has a particle size of 10 to 40 nm, preferably 20 to 30 nm. Nanogels are already widely commercially available, and these commercially available products may be used.
　本発明の実施形態で使用されるナノゲルは、ワクチンが負に帯電する鼻粘膜表面へ侵入できるように、正電荷を有する官能基、例えばアミノ基を導入したナノゲルである。アミノ基のナノゲルへの導入の方法としては、アミノ基を付加したコレステロールプルラン（CHPNH2）を用いる方法を挙げることができる。具体的には、減圧乾燥したCHPをジメチルスルホキシド（DMSO）に溶解し、これに1-1’カルボニルジイミダゾールを窒素気流下に加え数時間、室温で反応させる。その反応溶液にエチレンジアミンを徐々に添加し、数時間から数十時間程度攪拌する。得られた反応溶液を蒸留水に対して、数日間透析する。透析後の反応溶液を凍結乾燥し、乳白色の固体を得る。エチレンジアミンの置換度は元素分析やH-NMRなどを用いて評価することができる。 The nanogel used in the embodiment of the present invention is a nanogel into which a functional group having a positive charge, for example, an amino group has been introduced so that the vaccine can enter the surface of the negatively charged nasal mucosa. Examples of a method for introducing an amino group into the nanogel include a method using cholesterol pullulan (CHPNH 2 ) to which an amino group is added. Specifically, CHP dried under reduced pressure is dissolved in dimethyl sulfoxide (DMSO), and 1-1'carbonyldiimidazole is added thereto under a nitrogen stream, and the mixture is reacted for several hours at room temperature. Ethylenediamine is gradually added to the reaction solution and stirred for several hours to several tens of hours. The obtained reaction solution is dialyzed against distilled water for several days. The reaction solution after dialysis is freeze-dried to obtain a milky white solid. The degree of substitution of ethylenediamine can be evaluated using elemental analysis, H-NMR and the like.
　ワクチン抗原は、特に限定されず、ワクチン製剤の用途に応じて任意に選択することができる。とりわけ、本発明にかかるワクチン製剤は、細胞性免疫を効率よく誘導することができるため、疾患等の予防または治療上、細胞性免疫系の賦活化のための使用に大変適している。そのような疾患として、あえて例示すれば、成人に対する有効なワクチンが存在しない結核、ワクチン自体が存在しない無莢膜型インフルエンザ菌（NTHi）、RSV（respiratory syncytial virus）感染症もしくはHSV（herpes simplex virus）感染症またはその治療上細胞性免疫の誘導が重要と考えられるHPV（human papilloma virus）感染症およびその感染で発症する子宮頸がんなどが挙げられる。 The vaccine antigen is not particularly limited, and can be arbitrarily selected according to the use of the vaccine preparation. In particular, the vaccine preparation according to the present invention can induce cell-mediated immunity efficiently, and is therefore very suitable for use in activating the cellular immune system in preventing or treating diseases and the like. Examples of such diseases are tuberculosis, for which there is no effective vaccine for adults, tuberculosis-free Haemophilus influenzae (NTHi), RSV (respiratory syncytial virus) infection, or HSV (herpes virus simple virus), for which there is no effective vaccine for adults. ) Infectious diseases or HPV (human papilloma virus) infections, for which induction of cell-mediated immunity is considered to be important for the treatment thereof, and cervical cancer caused by the infection.
　結核のワクチン抗原としては、特に限定はしないが、例えば、結核菌（Mycobacterium tuberculosis）由来のAg85B（Rv1886）遺伝子産物、ESAT6（Rv3875）遺伝子産物、Rv2660遺伝子産物、Rv2608遺伝子産物、Rv3619遺伝子産物、Rv3620遺伝子産物、Rv1813遺伝子産物、MTB32A（Rv0125）遺伝子産物、MTB39A（Rv1196）遺伝子産物、MVA85A遺伝子産物またはRv0288遺伝子産物の全体もしくはその一部、あるいは、これらのタンパク質から選択される複数の融合タンパク質（例えば、ESAT6-Rv2660-Rv0288遺伝子産物のキメラタンパク質）であってもよい。
　無莢膜型インフルエンザ菌（NTHi）のワクチン抗原としては、D15、P1、P2、P4、P5、P6、Hmw/hia、Hap、Protein E、ProteinF、ProteinD、Pil A、NucA、HtrA、OMP26、PCP、TbpBまたはLOSの全体もしくはその一部、あるいは、これらのタンパク質から選択される複数の融合タンパク質であってもよい。
　RSVのワクチン抗原としては、特に限定はしないが、例えば、RSV由来のF蛋白（fusion protein）またはSH蛋白全体もしくはその一部、あるいは、これらのタンパク質から選択される複数の融合タンパク質であってもよい。
　HSVのワクチン抗原としては、特に限定はしないが、例えば、HSV由来のgD遺伝子産物、gB遺伝子産物、gC遺伝子産物、gE遺伝子産物、カプシド蛋白UL19、Tegment蛋白UL47またはgG遺伝子産物の全体もしくはその一部、あるいは、これらのタンパク質から選択される複数の融合タンパク質であってもよい。
　HPVのワクチン抗原としては、特に限定はしないが、例えば、HPV由来のE6遺伝子産物、特にがん抑制遺伝子産物P53のE6結合箇所の変異もしくは欠損産物、HPV由来のE7遺伝子産物、特にがん抑制遺伝子産物RbのE7結合箇所の変異もしくは欠損産物などであって、より具体的にはHPV6 E7 (23-27欠損)、HPV11 E7 (23-27欠損)、HPV16 E7（D21G, C24G, E26G変異）もしくはHPV16 E7(21-24欠損)、HPV18 E7（24-27欠損）、HPV31 E7（22-26欠損）、HPV33 E7（22-26欠損）、HPV45 E7（26-30欠損）、HPV52 E7（22-26欠損）またはHPV52 E7（22-26欠損）もしくはHPV58 E7（22-26欠損）の全体もしくはその一部、あるいは、これらのタンパク質から選択される複数の融合タンパク質であってもよい。 Examples of the vaccine antigen for tuberculosis include, but are not limited to, Ag85B (Rv1886) gene product, ESAT6 (Rv3875) gene product, Rv2660 gene product, Rv2608 gene product, Rv3619 gene product, and Rv3620 derived from Mycobacterium tuberculosis. Gene product, Rv1813 gene product, MTB32A (Rv0125) gene product, MTB39A (Rv1196) gene product, MVA85A gene product or all or part of Rv0288 gene product, or a plurality of fusion proteins selected from these proteins (for example, , ESAT6-Rv2660-Rv0288 gene product).
Examples of vaccine antigens for non-capsulated Haemophilus influenzae (NTHi) include D15, P1, P2, P4, P5, P6, Hmw / hia, Hap, Protein E, ProteinF, ProteinD, Pil A, NucA, HtrA, OMP26, PCP , TbpB or LOS, or a part thereof, or a plurality of fusion proteins selected from these proteins.
The RSV vaccine antigen is not particularly limited. For example, RSV-derived F protein (fusion protein) or whole or a part of SH protein, or a plurality of fusion proteins selected from these proteins may be used. Good.
HSV vaccine antigens are not particularly limited, but include, for example, HSV-derived gD gene product, gB gene product, gC gene product, gE gene product, capsid protein UL19, Tegment protein UL47 or the whole or one of gG gene products. Or a plurality of fusion proteins selected from these proteins.
Examples of the HPV vaccine antigen include, but are not limited to, HPV-derived E6 gene product, particularly a mutation or deletion product at the E6 binding site of tumor suppressor gene product P53, HPV-derived E7 gene product, particularly cancer suppression. Mutations or deletions at the E7 binding site of the gene product Rb, more specifically HPV6 E7 (23-27 deletion), HPV11 E7 (23-27 deletion), HPV16 E7 (D21G, C24G, E26G mutation) Or HPV16 E7 (21-24 deficiency), HPV18 E7 (24-27 deficiency), HPV31 E7 (22-26 deficiency), HPV33 E7 (22-26 deficiency), HPV45 E7 (26-30 deficiency), HPV52 E7 (22 -26 deficient) or HPV52 E7 (22-26 deficient) or HPV58 E7 (22-26 deficient), or a part thereof, or a plurality of fusion proteins selected from these proteins.
　本発明の実施形態で使用されるアジュバントとは、抗原性補強剤または免疫賦活化剤などと称されるものと同義で、当該分野において、これらの剤の通常の使用目的に用いられるものである。本発明の実施形態で使用されるアジュバントの有効成分は、特に限定はしないが、例えば、STING（stimulator of interferon genes）を活性化するSTINGリガンド（例えばcGAMP、cyclic-di AMP、cyclic-di GMP、cyclic-di CMP、cyclic-di UMPまたはcyclic-di IMPなどの環状ジヌクレオチドやDMXAA（5,6-dimethylXAA (xanthenone-4- acetic acid)、VadimezanまたはASA404）などのキサンテノン（Xanthenone）誘導体）、polyICまたはCpG ODNなどを挙げることができる。当該アジュバントは、さらに、医薬上許容される担体やその他の成分（例えば、安定化剤、pH調整剤、保存剤、防腐剤および緩衝剤など）を含んでいてもよい。医薬上許容される担体およびその他の成分は、ワクチン投与される動物の健康に悪影響を及ぼさない物質であることが必要である。 The adjuvant used in the embodiment of the present invention is synonymous with what is called an antigenic enhancer or an immunopotentiator, and is used in the art for the usual purpose of using these agents. . The active ingredient of the adjuvant used in the embodiment of the present invention is not particularly limited. For example, STING ligands that activate STING (stimulator of interferon genes) (eg, cGAMP, cyclic-di AMP, cyclic-di GMP, Cyclic dinucleotides such as cyclic-di CMP, cyclic-di UMP or cyclic-di IMP, xanthenone (Xanthenone) derivatives such as DMXAA (5,6-dimethylXAA (xanthenone-4- acetic acid), Vadimezan or ASA404), polyIC Alternatively, CpG-ODN and the like can be mentioned. The adjuvant may further contain a pharmaceutically acceptable carrier and other components (for example, a stabilizer, a pH adjuster, a preservative, a preservative, a buffer and the like). Pharmaceutically acceptable carriers and other ingredients need to be substances that do not adversely affect the health of the animal being vaccinated.
　ナノゲル、ワクチン抗原およびアジュバント（または、アジュバントの有効成分、以下同じ）の複合体は、ナノゲル、ワクチン抗原およびアジュバントを共存させ、相互作用させ、抗原とアジュバントをナノゲル内に取り込ませることにより作製することができる。このとき、ナノゲルとワクチン抗原、ナノゲルとアジュバントの混合比は、特に限定されず、当業者であれば予備的な実験により容易に決定することができる。あえて目安を挙げるとすれば、ワクチン抗原：ナノゲルが、モル比で、例えば、0.1:10、1:5、1:2または1:1程度である。また、アジュバントの含量は、ワクチン100重量％に対して、0.01重量％～99.99重量％程度含まれていてもよく、抗原1重量に対し、例えば、0.01重量～10重量程度であってもよい。 The complex of the nanogel, the vaccine antigen and the adjuvant (or the active ingredient of the adjuvant, the same applies hereinafter) is prepared by allowing the nanogel, the vaccine antigen and the adjuvant to coexist and interact, and incorporating the antigen and the adjuvant into the nanogel. Can be. At this time, the mixing ratio of the nanogel and the vaccine antigen, and the mixing ratio of the nanogel and the adjuvant are not particularly limited, and can be easily determined by those skilled in the art through preliminary experiments. As a rough guide, the vaccine antigen: nanogel ratio is, for example, about 0.1: 10, 1: 5, 1: 2 or 1: 1 in molar ratio. The content of the adjuvant may be about 0.01% to 99.99% by weight based on 100% by weight of the vaccine, and may be, for example, about 0.01% to 10% by weight based on 1% of the antigen.
　ナノゲル、ワクチン抗原およびアジュバントの複合体の形成は、ナノゲル、ワクチン抗原およびアジュバントを混合し、4～50℃、例えば、40℃で、30分～48時間、例えば、1時間程度静置して実施することができる。ナノゲル、ワクチン抗原およびアジュバントの複合体形成に使用するバッファーは、特に限定されず、あえて例示するならば、Tris-HCl緩衝液などが挙げられる。 The formation of the complex of the nanogel, the vaccine antigen and the adjuvant is performed by mixing the nanogel, the vaccine antigen and the adjuvant, and allowing the mixture to stand at 4 to 50 ° C, for example, 40 ° C, for 30 minutes to 48 hours, for example, about 1 hour. can do. The buffer used for forming the complex of the nanogel, the vaccine antigen and the adjuvant is not particularly limited, and for example, a Tris-HCl buffer and the like can be mentioned.
　本発明のワクチン製剤は、組成物（本発明のワクチン組成物）として、薬理学上許容された添加剤を含んでいてもよい。本発明のワクチン製剤は経鼻投与に適したものであり、剤形としても、経鼻投与が可能な形体が望ましく、液体製剤（点鼻剤および注射剤など）などが挙げられる。
　本発明のワクチン製剤が液体製剤の場合、有効成分を必要に応じて塩酸、水酸化ナトリウム、乳糖、乳酸、ナトリウム、リン酸一水素ナトリウムおよびリン酸二水素ナトリウムなどのｐＨ調整剤、塩化ナトリウムおよびブドウ糖などの等張化剤と共に製剤用蒸留水に溶解し、無菌濾過してアンプルに充填するか、さらに、マンニトール、デキストリン、シクロデキストリンおよびゼラチンなどを加えて真空凍結乾燥し、用事溶解型の製剤としてもよい。当該液体製剤には、薬学的に許容できる公知の安定剤、防腐剤、酸化防止剤等が含まれていても良く、安定剤としては、例えば、ゼラチン、デキストランおよびソルビトール等が、防腐剤としては、例えば、チメロサールおよびβプロピオラクトン等が、酸化防止剤としては、例えば、αトコフェロール等が挙げられる。 The vaccine preparation of the present invention may contain a pharmacologically acceptable additive as a composition (the vaccine composition of the present invention). The vaccine preparation of the present invention is suitable for nasal administration, and is desirably in a form capable of nasal administration, and examples thereof include liquid preparations (nasal drops and injections).
When the vaccine preparation of the present invention is a liquid preparation, the active ingredient is optionally adjusted with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate and sodium dihydrogen phosphate, sodium chloride and It is dissolved in distilled water for preparation together with an isotonic agent such as glucose, and then sterile-filtered and filled into ampoules, or mannitol, dextrin, cyclodextrin, gelatin, etc. are added and freeze-dried in vacuum, and the working-soluble preparation is prepared. It may be. The liquid formulation may contain known pharmaceutically acceptable stabilizers, preservatives, antioxidants, and the like.Examples of the stabilizers include gelatin, dextran, sorbitol, and the like. For example, thimerosal and β-propiolactone, and as the antioxidant, for example, α-tocopherol and the like.
　本発明の第２の実施形態は、ナノゲル、ワクチン抗原およびアジュバントの複合体を含むワクチン製剤（第１の実施形態）を患者に経鼻投与することを含む、疾患の予防および／または治療方法である。
　第２の実施形態の治療または予防の対象疾患は、使用するワクチン抗原に依存し、特に限定はされず、病原体による感染症（例えば、結核、HSVおよびRSVなど）の他、がん（例えば、子宮頸がん）などであってもよく、細胞性免疫によって、治癒等が期待される疾患の全てを含む。
　本発明のワクチン製剤は、鼻粘膜を介して投与してもよい。その方法としては、例えば、鼻粘膜への噴霧、塗布、滴下等により鼻腔内へ投与する方法が挙げられる。 A second embodiment of the present invention is directed to a method for preventing and / or treating a disease, comprising intranasally administering to a patient a vaccine formulation (first embodiment) comprising a complex of a nanogel, a vaccine antigen and an adjuvant. is there.
The disease to be treated or prevented in the second embodiment depends on the vaccine antigen to be used and is not particularly limited. In addition to infectious diseases caused by pathogens (eg, tuberculosis, HSV and RSV, etc.), cancers (eg, Cervical cancer), and includes all diseases that are expected to be cured by cellular immunity.
The vaccine formulation of the present invention may be administered via the nasal mucosa. Examples of the method include a method in which the drug is administered into the nasal cavity by spraying, applying, dripping, or the like on the nasal mucosa.
　粘膜ワクチン製剤の投与量は、投与対象の年齢や体重等により適宜決定することができるが、薬学的に有効な量のワクチン抗原を含む。薬学的に有効な量とは、そのワクチン抗原に対する免疫反応を誘導するのに必要な抗原量をいう。例えば、１回のワクチン抗原投与量数μg～数10mgで1日1回～数回投与し、1～数週間間隔でトータル数回、例えば1～5回程度投与すればよい。 投 与 The dose of the mucosal vaccine preparation can be appropriately determined depending on the age, weight, and the like of the administration subject, and includes a pharmaceutically effective amount of the vaccine antigen. A pharmaceutically effective amount refers to that amount of antigen required to induce an immune response to the vaccine antigen. For example, the vaccine antigen may be administered once to several times a day at a dose of several μg to several tens mg, and may be administered several times at intervals of one to several weeks, for example, about 1 to 5 times.
　本明細書において引用されたすべての文献の開示内容は、全体として明細書に参照により組み込まれる。また、本明細書全体において、単数形の「a」、「an」、および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものを含むものとする。
　以下に実施例を示してさらに本発明の説明を行うが、実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 The disclosures of all documents cited herein are hereby incorporated by reference in their entirety. Also, throughout this specification, the use of the words "a", "an", and "the" in the singular includes the singular as well as the plural unless the context clearly indicates otherwise. Shall be included.
Hereinafter, the present invention will be further described with reference to examples. However, the examples are merely examples of the embodiments of the present invention, and do not limit the scope of the present invention.
方法
１．結核菌ワクチン
１－１．抗原タンパク質の調製
　結核菌（ATCC25618）由来のAg85B遺伝子（987bp）（配列番号１）を人工合成し、His-Tag配列の遺伝子を持つpET-20b(+) ベクター (Novagen)のEcoRI-HinIII (タカラバイオ社)サイトに挿入した。作製した発現ベクターを定法にてRosetta2 (DE3 )pLysS-大腸菌に形質転換した。得られた形質転換体を100 μg/mL ampicillin および 34 μg/mL chloramphenicolを含む培地中、37℃で、OD600nmが0.5-0.8になるまで培養した。その後1.0 mM isopropyl β-D-1-thiogalactopyranoside（和光純薬）を加えて4時間培養した。培養した大腸菌を遠心分離（5,000rpm、15分）で回収した。回収した大腸菌を10 mM imidazole とprotease inhibitor（Roche Diagnostics）を含む溶液で洗浄し、タンパク質を20mM Tris-HCl、500mM NaCl、10mM imidazoleおよび6 M ureaを含む吸着緩衝液で抽出した。抽出したタンパク質分画をnickel affinity カラム（GE Healthcare Bio-Sciences 社）にチャージして吸着緩衝液でOD280nmが0.01以下になるまで洗浄し、20mM Tris-HCl、500mM NaCl、500 mM imidazoleおよび6 M ureaを含む溶液で、タンパク質を溶出した。次いで、溶出液をアミコンで濃縮し、6M-Urea PBSで平衡化したSephacryl S-100 カラム（GE Healthcare Bio-Sciences社）でゲルろ過し、Ag85B分画を回収し、4M-Urea PBS、2M-Urea PBS、１M-Urea PBS、PBSと段階的に透析して、native Ag85Bを調製した。12 Lの大腸菌培養で50 mgのAg85B（配列番号２）が回収し、純度はSDS-PAGEにて95%であった。 Method 1. Mycobacterium tuberculosis vaccine 1-1. Preparation of Antigen Protein An Ag85B gene (987 bp) (SEQ ID NO: 1) derived from Mycobacterium tuberculosis (ATCC25618) was artificially synthesized, and a pET-20b (+) vector (Novagen) vector containing a His-Tag sequence gene (Novagen) EcoRI-HinIII (Takara) (Bio Inc.) site. The prepared expression vector was transformed into Rosetta2 (DE3) pLysS-E. Coli by a conventional method. The obtained transformant was cultured in a medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol at 37 ° C. until the OD600nm reached 0.5-0.8. Thereafter, 1.0 mM isopropyl β-D-1-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added, and the cells were cultured for 4 hours. The cultured E. coli was collected by centrifugation (5,000 rpm, 15 minutes). The collected E. coli was washed with a solution containing 10 mM imidazole and protease inhibitor (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole and 6 M urea. The extracted protein fraction is charged to a nickel affinity column (GE Healthcare Bio-Sciences), washed with an adsorption buffer until the OD 280 nm becomes 0.01 or less, 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole and 6 M urea. The protein was eluted with a solution containing Next, the eluate was concentrated with Amicon, and subjected to gel filtration with a Sephacryl S-100 column (GE Healthcare Bio-Sciences) equilibrated with 6M-Urea PBS, and the Ag85B fraction was collected, and 4M-Urea PBS, 2M- Urea PBS, 1M-Urea PBS and PBS were dialyzed stepwise to prepare native Ag85B. 50 mg of Ag85B (SEQ ID NO: 2) was recovered in a 12 L culture of E. coli, and the purity was 95% by SDS-PAGE.
１－２．抗原のナノゲル化（ワクチンの調製）
　cCHPナノゲルは、既報（非特許文献２）の方法に従って調製した。
　調製したcCHPナノゲルと精製したAg85Bタンパク質、を分子比1：1で混合し、さらに、アジュバントとして3種類のSTINGリガンド（cyclic-di-GMP、cyclic-di-AMPおよびcGAMP）を各々加えた後、40℃のヒートブロックで1時間インキュベーションした。
　また、cCHPナノゲルとキメラ精製タンパク質（ESAT6- Rv2660c-Rv0288）（アミノ酸配列：配列番号８、核酸配列：配列番号９）を分子比1：1で混合し、さらに、粘膜アジュバントとしてSTINGリガンド（cyclic-di-AMP）を加えたのち、40℃のヒートブロックで1時間インキュベーションした。 1-2. Nanogelation of antigen (preparation of vaccine)
The cCHP nanogel was prepared according to the method described previously (Non-Patent Document 2).
After mixing the prepared cCHP nanogel and the purified Ag85B protein at a molecular ratio of 1: 1 and further adding three types of STING ligands (cyclic-di-GMP, cyclic-di-AMP and cGAMP) as adjuvants, Incubated for 1 hour in a 40 ° C. heat block.
In addition, cCHP nanogel and chimeric purified protein (ESAT6-Rv2660c-Rv0288) (amino acid sequence: SEQ ID NO: 8, nucleic acid sequence: SEQ ID NO: 9) were mixed at a molecular ratio of 1: 1, and further, as a mucosal adjuvant, STING ligand (cyclic-cyclic) was used. After adding di-AMP), the mixture was incubated in a heat block at 40 ° C. for 1 hour.
１－３．マウスへの経鼻免疫
　cCHP-Ag85B+STINGリガンド混合溶液を、Balb/cマウスの7週齢メスに経鼻的に投与した。投与抗原量は、1匹一回あたりAg85Bタンパク量に換算して10 μgを投与した。また、STINGリガンドは、1匹一回あたり1 μg～10 μgの範囲で調製し投与した。経鼻免疫は1週間隔で計3回実施した。
　また、cCHP-キメラ+STINGリガンド溶液を、Balb/cマウスの7週齢メスに経鼻的に投与した。投与抗原量は、1匹一回あたりキメラタンパク量として10 μgを、STINGリガンドとして10 μgを投与した。経鼻免疫は1週間隔で計3回実施した。 1-3. Nasal Immunization to Mice A mixed solution of cCHP-Ag85B + STING ligand was intranasally administered to a 7-week-old female Balb / c mouse. The amount of antigen to be administered was 10 μg in terms of the amount of Ag85B protein per animal at one time. The STING ligand was prepared and administered in the range of 1 μg to 10 μg per animal. Nasal immunization was performed three times at weekly intervals.
Further, the cCHP-chimera + STING ligand solution was intranasally administered to a 7-week-old female Balb / c mouse. The amount of antigen to be administered was 10 μg as chimeric protein and 10 μg as STING ligand per animal. Nasal immunization was performed three times at weekly intervals.
１－４．抗原特異的T細胞の精製およびカウント
（１）Ag85B抗原
　ワクチンの最終投与から2週目に抗原特異的なTh1細胞（IFNγ産生細胞）またはTh17細胞（IL-17産生細胞）をELISPOT法でカウントした。全身性の免疫応答は脾臓で、粘膜面における免疫応答は肺組織に生成された抗原特異的T細胞で評価した。
　マウスを安楽死させたのち、肺および脾臓を摘出し細胞懸濁液を調製した。調製した細胞懸濁液から、MACS sytem（MiltenyiBiotec）を用いて、CD4陽性T細胞を精製した。一方、未免疫のマウス脾臓から、CD90.2陰性細胞を同様に精製し抗原提示細胞とした。CD4陽性T細胞およびガンマ線照射した抗原提示細胞を、精製Ag85B抗原刺激下で48～72時間共培養した。ここで、培養ウェルの底には、抗IFNγまたは抗IL-17抗体をキャプチャー抗体として予め吸着させた。
　培養上清および細胞を除去し、ウェルを洗浄後、ビオチン標識の抗IFNγ抗体または抗IL-17抗体を加えて、室温にて2時間反応させた。その後、ウェルを洗浄し、ストレプトアビジンHRPを反応させ、洗浄後にHRPの基質である3-Amino-9-ethylcarbazole（AEC）を添加し発色させ、抗原特異的Th1細胞またはTh17細胞をスポットとして検出した。スポット数はエリスポットカウンターにより計測した。 1-4. Purification and counting of antigen-specific T cells (1) Ag85B antigen Two weeks after the final administration of the vaccine, antigen-specific Th1 cells (IFNγ-producing cells) or Th17 cells (IL-17-producing cells) were counted by the ELISPOT method. . The systemic immune response was evaluated in the spleen, and the mucosal immune response was evaluated using antigen-specific T cells generated in lung tissue.
After euthanizing the mouse, the lung and spleen were excised to prepare a cell suspension. CD4-positive T cells were purified from the prepared cell suspension using MACS sytem (MiltenyiBiotec). On the other hand, CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells. CD4-positive T cells and gamma-irradiated antigen-presenting cells were co-cultured under purified Ag85B antigen stimulation for 48-72 hours. Here, an anti-IFNγ or anti-IL-17 antibody was previously adsorbed to the bottom of the culture well as a capture antibody.
After removing the culture supernatant and the cells and washing the wells, a biotin-labeled anti-IFNγ antibody or anti-IL-17 antibody was added, and reacted at room temperature for 2 hours. After that, the wells were washed and reacted with streptavidin HRP. After washing, HRP substrate 3-Amino-9-ethylcarbazole (AEC) was added to develop color, and antigen-specific Th1 cells or Th17 cells were detected as spots. . The number of spots was measured using an ELISPOT counter.
（２）ESAT6-Rv2660c-Rv0288キメラ抗原
　最終投与から2週で抗原特異的なTh1細胞（IFNγ産生細胞）をELISPOT法でカウントした。全身性の免疫応答は脾臓で、粘膜面における免疫応答は肺組織に生成された抗原特異的T細胞で評価した。
　マウスを安楽死させたのち、肺および脾臓を摘出し細胞懸濁液を調整した。そこからマグネティックビーズを用いて、CD4陽性のT細胞を精製した。一方、未免疫のマウス脾臓から、CD90.2陰性細胞を同様に精製し抗原提示細胞とした。CD4陽性T細胞およびガンマ線照射した抗原提示細胞を、精製キメラ抗原またはリコンビナントESAT6（アブカム社）刺激下で48～72時間共培養した。培養ウェルの底に、抗IFNγをキャプチャーとして敷き産生細胞を検出した。
　培養上清を除き、ウェルを洗浄後、ビオチン標識の抗IFNγ抗体を反応させた。さらに洗浄後、ストレプトアビジンHRPを反応させ、洗浄後にHRPの基質である3-Amino-9-ethylcarbazole（AEC）を反応して発色させ、抗原特異的Th1をスポットとして検出した。スポットはエリスポットカウンターにより計測した。 (2) ESAT6-Rv2660c-Rv0288 chimeric antigen Two weeks after the last administration, antigen-specific Th1 cells (IFNγ-producing cells) were counted by the ELISPOT method. The systemic immune response was evaluated in the spleen, and the mucosal immune response was evaluated using antigen-specific T cells generated in lung tissue.
After the mouse was euthanized, the lung and spleen were removed and the cell suspension was prepared. From there, CD4 positive T cells were purified using magnetic beads. On the other hand, CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells. CD4-positive T cells and gamma-irradiated antigen presenting cells were co-cultured for 48-72 hours under the stimulation of purified chimeric antigen or recombinant ESAT6 (Abcam). At the bottom of the culture well, anti-IFNγ was used as a capture to detect production cells.
After removing the culture supernatant, the wells were washed and reacted with a biotin-labeled anti-IFNγ antibody. Further, after washing, streptavidin HRP was reacted, and after washing, 3-Amino-9-ethylcarbazole (AEC), a substrate of HRP, was reacted to develop color, and antigen-specific Th1 was detected as a spot. The spot was measured by an ELISPOT counter.
１－５．防御免疫効果の検討
（１）マウスへのワクチン投与
　マウスは、Balb/cの7週齢メスを用いた。ポジティブコントロールのBCGワクチンは、PBS溶液に懸濁してマウスに初回免疫時に1回皮下投与した。cCHP-Ag85B+cyclic-di-GMPの混合溶液は、1匹一回あたりAg85Bタンパク量として10 μgを1週間隔で計3回経鼻投与した。未免疫コントロールマウスには、PBSを1週おきに3回経鼻的におよび初回免疫時に1回皮下投与した。
（２）結核菌強毒株の経気道感染
　ワクチンの最終免疫から8週後に、結核菌強毒株Erdmanを1匹あたり100 CFU経気道感染させた。
（３）脾臓および肺組織中の結核菌数の計測
　感染後12週においてマウスを安楽死させ、肺および脾臓を摘出し、PBS中で組織を破砕して懸濁し、6つの希釈系列を調製してそれぞれ寒天培地に播種した。嫌気的な環境下で4週間培養し、コロニーを計測してそれぞれの組織中の結核菌数を算出した。 1-5. Examination of protective immunity (1) Vaccine administration to mice The mice were 7-week-old Balb / c females. The BCG vaccine as a positive control was suspended in a PBS solution and administered subcutaneously once to mice at the time of the first immunization. The mixed solution of cCHP-Ag85B + cyclic-di-GMP was nasally administered at a dose of 10 μg per animal once per week at an interval of 10 μg of Ag85B protein. Unimmunized control mice received PBS intranasally three times every other week and once subcutaneously at the time of the first immunization.
(2) Trans-respiratory tract infection with Mycobacterium tuberculosis virulent strain Eight weeks after the final immunization with the vaccine, Mycobacterium tuberculosis virulent strain Erdman was transinfected by 100 CFU per animal.
(3) Counting of Mycobacterium tuberculosis counts in spleen and lung tissues At 12 weeks after infection, mice were euthanized, lungs and spleen were excised, and tissues were crushed and suspended in PBS to prepare six dilution series. Each was seeded on an agar medium. The cells were cultured in an anaerobic environment for 4 weeks, the number of colonies was counted, and the number of M. tuberculosis in each tissue was calculated.
２．HPVワクチンの調製
２－１．抗原タンパク質の調製
　HPV16ウイルスのガン抑制遺伝子産物の3アミノ酸D21G、C24GおよびE26G変異E7（Van der Burg SH et.al. Vaccine 19:3652-3660, 2001）遺伝子(307bp)（配列番号３）を人工合成し、His-Tag配列の遺伝子を持つpET-20b(+) ベクター (Novagen)のEcoRI-HinIII (タカラバイオ社)サイトに挿入した。作製した発現ベクターを定法にてRosetta2 (DE3 )pLysS-大腸菌に形質転換した。得られた形質転換体を100 μg/mL ampicillin および 34 μg/mL chloramphenicolを含む培地中、37℃で、OD600nmが0.5-0.8になるまで培養した。その後1.0 mM isopropyl β-D-1-thiogalactopyranoside（和光純薬）を加えて4時間培養した。培養した大腸菌を遠心分離（5,000rpm、15分）で回収した。回収した大腸菌を10 mM imidazole とprotease inhibitor（Roche Diagnostics）を含む溶液で洗浄し、タンパク質を20mM Tris-HCl、500mM NaCl、10mM imidazoleおよび6 M ureaを含む吸着緩衝液で抽出した。抽出したタンパク質分画をnickel affinity カラム（GE Healthcare Bio-Sciences 社）にチャージして吸着緩衝液でOD280nmが0.01以下になるまで洗浄し、20mM Tris-HCl、500mM NaCl、500 mM imidazoleおよび6 M ureaを含む溶液で、タンパク質を溶出した。次いで、溶出液を6M Urea-PBS（0.15M NaCl）で透析し、同緩衝液で平衡化したDEAE-sepharoseカラム（GE Healthcare Bio-Sciences K.K）に吸着させ、0.5M NaCl-PBS-6 M Ureaを含む液で溶出した。この溶出液をアミコンで濃縮し、6M-Urea PBSで平衡化したSephacryl S-100 カラム(GE Healthcare Bio-Sciences社)でゲルろ過し、変異型E7分画を回収し、４M-Urea PBS、2M-Urea PBS、1M-Urea PBS、PBSと段階的に透析して、native 変異型E7（配列番号４）を調製した。12Lの大腸菌培養で60mgの変異型E7が回収し、純度はSDS-PAGEにて95%であった。 2. Preparation of HPV vaccine 2-1. Preparation of antigen protein The three amino acids D21G, C24G and E26G mutation E7 (Van der Burg SH et.al. Vaccine 19: 3652-3660, 2001) of the tumor suppressor gene product of HPV16 virus (307 bp) (SEQ ID NO: 3) were artificially generated. It was synthesized and inserted into EcoRI-HinIII (Takara Bio Inc.) site of a pET-20b (+) vector (Novagen) having a gene of His-Tag sequence. The prepared expression vector was transformed into Rosetta2 (DE3) pLysS-E. Coli by a conventional method. The obtained transformant was cultured in a medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol at 37 ° C. until the OD600nm reached 0.5-0.8. Thereafter, 1.0 mM isopropyl β-D-1-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added, and the cells were cultured for 4 hours. The cultured E. coli was collected by centrifugation (5,000 rpm, 15 minutes). The collected E. coli was washed with a solution containing 10 mM imidazole and protease inhibitor (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole and 6 M urea. The extracted protein fraction is charged to a nickel affinity column (GE Healthcare Bio-Sciences), washed with an adsorption buffer until the OD 280 nm becomes 0.01 or less, 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole and 6 M urea. The protein was eluted with a solution containing Subsequently, the eluate was dialyzed against 6 M Urea-PBS (0.15 M NaCl), adsorbed on a DEAE-sepharose column (GE Healthcare Bio-Sciences KK) equilibrated with the same buffer, and then 0.5 M NaCl-PBS-6 M Urea Eluted with a solution containing The eluate was concentrated with Amicon, gel-filtered through a Sephacryl S-100 column (GE Healthcare Bio-Sciences) equilibrated with 6M-Urea PBS, and the mutant E7 fraction was collected, and 4M-Urea PBS, 2M -Urea PBS, 1M-Urea PBS and PBS were dialyzed stepwise to prepare native mutant E7 (SEQ ID NO: 4). 60 mg of mutant E7 was recovered in 12 L of E. coli culture, and the purity was 95% by SDS-PAGE.
２－２．抗原のナノゲル化（ワクチンの調製）
　cCHPナノゲルは、既報（非特許文献２）の方法に従って調製した。
　調製したcCHPナノゲルと精製した変異型E7タンパク質を分子比1：1で混合し、さらに、アジュバントとしてcyclic-di-AMPのみ、STINGリガンド3種（cyclic-di-GMP, cyclic-di-AMP, cGAMP）、または、poly I:C、CpG ODN K3型またはD35型をそれぞれ加えたのち、40℃のヒートブロックで1時間インキュベーションした。 2-2. Nanogelation of antigen (preparation of vaccine)
The cCHP nanogel was prepared according to the method described previously (Non-Patent Document 2).
The prepared cCHP nanogel and the purified mutant E7 protein are mixed at a molecular ratio of 1: 1. Further, only adjuvant cyclic-di-AMP and three kinds of STING ligands (cyclic-di-GMP, cyclic-di-AMP, cGAMP ) Or poly I: C, CpG ODN K3 or D35, respectively, and then incubated for 1 hour in a heat block at 40 ° C.
２－３．マウスへの経鼻免疫
　cCHP-変異E7+各粘膜アジュバントの混合溶液を、Balb/cマウスの7週齢メスに経鼻的に投与した。投与抗原量は、1匹一回あたり変異型E7タンパク量に換算して10 μgを投与した。また、各粘膜アジュバントは、1匹一回あたり5 μgまたは10μg投与した。経鼻免疫は1週間隔で計3回実施した。 2-3. Nasal Immunization to Mice A mixed solution of cCHP-mutant E7 and each mucosal adjuvant was intranasally administered to 7-week-old female Balb / c mice. The amount of the antigen to be administered was 10 μg in terms of the amount of mutant E7 protein per animal. Each mucosal adjuvant was administered at 5 μg or 10 μg per animal. Nasal immunization was performed three times at weekly intervals.
２－４．抗原特異的T細胞の精製およびカウント
（１）cyclic-di-AMPをアジュバントとした場合
　ワクチンの最終投与から1週目に抗原特異的なCTL細胞（グランザイムB産生細胞）またはTh1細胞（IFNγ産生細胞）をELISPOT法でカウントした。全身性の免疫応答は脾臓で、生殖器粘膜における免疫応答は子宮頸部に誘導された抗原特異的T細胞で評価した。
　マウスを安楽死させたのち、脾臓および子宮頸部を摘出し細胞懸濁液を調製した。調製した細胞懸濁液から、MACS sytem（MiltenyiBiotec）を用いて、T細胞（CD90.2陽性）を精製した。一方、未免疫のマウス脾臓から、CD90.2陰性細胞を同様に精製し抗原提示細胞とした。精製したT細胞およびガンマ線照射した抗原提示細胞を、精製変異E7抗原刺激下で48～72時間共培養した。ここで、培養ウェルの底には、抗グランザイムB抗体または抗IFNγ抗体をキャプチャー抗体として予め吸着させた。
　培養上清および細胞を除去し、ウェルを洗浄後、ビオチン標識の抗グランザイムB抗体または抗IFNγ抗体を加えて、室温にて2時間反応させた。その後、ウェルを洗浄し、ストレプトアビジンHRPを反応させ、洗浄後にHRPの基質である3-Amino-9-ethylcarbazole（AEC）を添加し発色させ、抗原特異的CTL細胞またはTh1細胞をスポットとして検出した。スポット数はエリスポットカウンターにより計測した。 2-4. Purification and counting of antigen-specific T cells (1) When cyclic-di-AMP was used as adjuvant One week after the final administration of the vaccine, antigen-specific CTL cells (granzyme B-producing cells) or Th1 cells (IFNγ-producing cells) ) Were counted by the ELISPOT method. The systemic immune response was assessed in the spleen, and the immune response in the genital mucosa was assessed with antigen-specific T cells induced in the cervix.
After the mouse was euthanized, the spleen and the cervix were removed to prepare a cell suspension. From the prepared cell suspension, T cells (CD90.2 positive) were purified using MACS sytem (MiltenyiBiotec). On the other hand, CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells. Purified T cells and gamma-irradiated antigen presenting cells were co-cultured under purified mutant E7 antigen stimulation for 48-72 hours. Here, an anti-granzyme B antibody or an anti-IFNγ antibody was previously adsorbed to the bottom of the culture well as a capture antibody.
After removing the culture supernatant and the cells and washing the wells, a biotin-labeled anti-granzyme B antibody or anti-IFNγ antibody was added, and the mixture was reacted at room temperature for 2 hours. Then, the wells were washed and reacted with streptavidin HRP. After washing, HRP substrate 3-Amino-9-ethylcarbazole (AEC) was added to develop color, and antigen-specific CTL cells or Th1 cells were detected as spots. . The number of spots was measured using an ELISPOT counter.
（２）STINGリガンド3種（cyclic-di-GMP, cyclic-di-AMP, cGAMP）をアジュバントとした場合
　最終投与から1週で、子宮頸部における抗原特異的なTh1細胞（IFNγ産生細胞）およびCTL（グランザイムB産生細胞）をELISPOT法でカウントした。マウスを安楽死させたのち、子宮頸部を摘出し細胞懸濁液を調整した。そこからマグネティックビーズを用いて、T細胞（CD90.2陽性）を精製した。一方、未免疫のマウス脾臓から、CD90.2陰性細胞を同様に精製し抗原提示細胞とした。精製T細胞およびガンマ線照射した抗原提示細胞を、精製変異E7抗原刺激下で48～72時間共培養した。培養ウェルの底に、抗IFNγ抗体または抗グランザイムB抗体をキャプチャーとして敷き産生細胞を検出した。
　培養上清を除き、ウェルを洗浄後、ビオチン標識の抗IFNγ抗体または抗グランザイムB抗体を反応させた。さらに洗浄後、ストレプトアビジンHRPを反応させ、洗浄後にHRPの基質であるAECを反応して発色させ、抗原特異的Th1またはCTLをスポットとして検出した。スポットはエリスポットカウンターにより計測した。 (2) When three kinds of STING ligands (cyclic-di-GMP, cyclic-di-AMP, cGAMP) were used as adjuvant One week after the last administration, antigen-specific Th1 cells (IFNγ-producing cells) in the cervix and CTLs (granzyme B-producing cells) were counted by the ELISPOT method. After the mice were euthanized, the cervix was removed and a cell suspension was prepared. From there, T cells (CD90.2 positive) were purified using magnetic beads. On the other hand, CD90.2-negative cells were similarly purified from unimmunized mouse spleens and used as antigen-presenting cells. Purified T cells and gamma-irradiated antigen-presenting cells were co-cultured for 48-72 hours under the stimulation of purified mutant E7 antigen. An anti-IFNγ antibody or an anti-granzyme B antibody was laid on the bottom of the culture well as a capture to detect production cells.
After removing the culture supernatant, the wells were washed and reacted with a biotin-labeled anti-IFNγ antibody or anti-granzyme B antibody. Further, after washing, streptavidin HRP was reacted, and after washing, AEC which is a substrate of HRP was reacted to develop color, and antigen-specific Th1 or CTL was detected as a spot. The spot was measured by an ELISPOT counter.
３．RSVワクチンの調製
３－１．抗原タンパク質の調製
　RSVウイルスのSHペプチド（配列番号５）にPspAをリンカー(GGGGS)（配列番号７）を介し3つ繰り返したDNA配列を人工合成し(1172bp)、制限酵素EcoRV とNotI (タカラバイオ社)を用いてHis-Tag配列の遺伝子を持つpET-20b(+) ベクター (Novagen)にインサートした。このプラスミドを定法にてRosetta2 (DE3 )pLysS-大腸菌に形質転換した。この大腸菌を100 μg/mL ampicillin および 34 μg /mL chloramphenicolを含む培地で、37℃でOD600nmが0.5-0.8になるまで培養した。その後、1.0 mM isopropyl β-D-1-thiogalactopyranoside (和光純薬)を加えて4時間培養後、大腸菌を遠心分離(5000rpm, 15分)で回収した。菌を20 mM imidazole とprotease inhibitor (Roche Diagnostics)を含む液で洗い、タンパク質を20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazoleを含む吸着緩衝液で抽出した。抽出液に80%飽和になるよう飽和硫酸アンモニウム溶液を加え、硫安沈殿をおこなった。沈殿物を遠心回収し、抽出時と同様の緩衝液を外液とし透析した。透析後の液をnickel affinity カラム (GE Healthcare Bio-Sciences 社)にチャージして吸着緩衝液でOD280nmが0.01以下になるまで洗い、20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazoleを含む液で溶出させた。この溶出液をアミコンで濃縮し、PBSで平衡化したSephadex G-100カラム(GE Healthcare Bio-Sciences社)でゲルろ過し、PspA-SH3分画を回収し、濃縮、精製した。20Lの大腸菌培養で70mgのPspA-SH3を回収し、純度はSDS-PAGEにて95%であった。 3. Preparation of RSV vaccine 3-1. Preparation of antigen protein An artificially synthesized DNA sequence (1172 bp) obtained by repeating PspA to the SH peptide (SEQ ID NO: 5) of RSV virus via a linker (GGGGS) (SEQ ID NO: 7) (1172 bp), restriction enzymes EcoRV and NotI (Takara Was inserted into a pET-20b (+) vector (Novagen) having a His-Tag sequence gene. This plasmid was transformed into Rosetta2 (DE3) pLysS-E. Coli by a conventional method. This Escherichia coli was cultured in a medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol at 37 ° C. until the OD600 nm reached 0.5-0.8. Thereafter, 1.0 mM isopropyl β-D-1-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added, and after culturing for 4 hours, Escherichia coli was recovered by centrifugation (5000 rpm, 15 minutes). The bacteria were washed with a solution containing 20 mM imidazole and protease inhibitor (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20 mM Tris-HCl, 500 mM NaCl, and 10 mM imidazole. A saturated ammonium sulfate solution was added to the extract to 80% saturation, and ammonium sulfate precipitation was performed. The precipitate was collected by centrifugation, and dialyzed using the same buffer solution as used for extraction as the external solution. The dialyzed solution is charged to a nickel affinity column (GE Healthcare Bio-Sciences), washed with an adsorption buffer until the OD 280 nm becomes 0.01 or less, and the solution containing 20 mM Tris-HCl, 500 mM NaCl, and 500 mM imidazole is used. Eluted. The eluate was concentrated with Amicon, gel-filtered through a Sephadex G-100 column (GE Healthcare Bio-Sciences) equilibrated with PBS, and the PspA-SH3 fraction was collected, concentrated and purified. 70 mg of PspA-SH3 was recovered by culturing 20 L of E. coli, and the purity was 95% by SDS-PAGE.
３－２．抗原のナノゲル化（ワクチンの調製）
　cCHPナノゲルとPspA-SH3精製タンパク質（配列番号６）を分子比1：1で混合し、さらに、粘膜アジュバントとしてcyclic-di-AMPを加えたのち、40℃のヒートブロックで1時間インキュベーションした。 3-2. Nanogelation of antigen (preparation of vaccine)
cCHP nanogel and PspA-SH3 purified protein (SEQ ID NO: 6) were mixed at a molecular ratio of 1: 1, and cyclic-di-AMP was added as a mucosal adjuvant, followed by incubation in a heat block at 40 ° C. for 1 hour.
３－３．マウスへの経鼻免疫
　cCHP-PspA-SH3+cyclic-di-AMPの混合溶液を、Balb/cマウスの7週齢メスに経鼻的に投与した。投与抗原量は、1匹一回あたりPspA-SH3タンパク量として10 μgを投与した。また、cyclic-di-AMPは、10ug投与した。経鼻免疫は1週間隔で3回ののち、4週あけて1回、さらに4週あけて1回の計5回おこなった。 3-3. Nasal Immunization to Mice A mixed solution of cCHP-PspA-SH3 + cyclic-di-AMP was intranasally administered to a 7-week-old female Balb / c mouse. The amount of antigen to be administered was 10 μg of PspA-SH3 protein per animal. Moreover, 10 ug of cyclic-di-AMP was administered. Nasal immunization was performed three times at weekly intervals, once every four weeks, and once every four weeks for a total of five times.
３－４．抗体価測定
　毎週、顎下静脈より100 μl程度採血し、15000 rpm, 4℃で遠心分離し、血清を回収した。
　PspAあるいはSH特異的血清中IgG抗体価の測定、IgGサブクラスの測定はELISA法で実施した。ELISA実施前日に、PspAもしくはBSA conjugate SHをPBSで1μg/mlとなるように希釈し、96wellプレート(Thermo scientific, 3355)に100 μlずつキャプチャーとして分注し、4℃で一晩インキュベートした。プレートウォッシャーを用いて300 μlの0.05 % Tween(nacalai tesque, 28353-85)含有PBS(PBS-T）で4回プレートを洗浄し、1 % BSA(nacalai tesque, 01863-48)含有PBS-Tを200 μL/well加え、室温で1時間インキュベートし、ウェルをブロッキングした。つぎに、プレートウォッシャーを用いて300 μlのPBS-Tで3回洗浄した。各サンプルを1 % BSA含有PBS-Tで28倍希釈したものをプレートの端のウェルに入れ、もう一端まで2倍段階希釈をおこない段階希釈系列を作製し、室温で2時間インキュベートを行った。ブランクは1 ％ BSA含有PBS-Tとした。インキュベート終了後、プレートウォッシャーを用いて300 μLのPBS-Tで4回プレートを洗浄した。続けて、Goat anti-Human IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3(Southern Biotech)の6種いずれかを1 %　BSA含有PBS-Tで4000倍希釈したものを100 μl/well加え、室温で1.5時間インキュベートした。その後、プレートウォッシャーを用いて300 μlのPBS-Tで4回プレートを洗浄した。TMB SubstrateとTMB Solution(seracare, 5120-0050)を等量混合したものを100 μl/well加え、30分発色反応させた後、2N H2SO4(nacalai tesque, 32520-55)を50 μl加え反応を停止させた。プレートリーダーでOD450の値を測定しlog2titerの値を算出した。カットオフ値はブランクウェルの平均値＋0.1とした。 3-4. Measurement of antibody titer Every week, about 100 μl of blood was collected from the submandibular vein, and centrifuged at 15000 rpm at 4 ° C. to collect serum.
The measurement of the IgG antibody titer in the PspA or SH-specific serum and the measurement of the IgG subclass were performed by ELISA. On the day before the ELISA, PspA or BSA conjugate SH was diluted to 1 μg / ml with PBS, dispensed into a 96-well plate (Thermo scientific, 3355) in 100 μl aliquots as captures, and incubated at 4 ° C. overnight. Wash the plate 4 times with 300 μl of PBS (PBS-T) containing 0.05% Tween (nacalai tesque, 28353-85) using a plate washer, and wash the PBS-T containing 1% BSA (nacalai tesque, 01863-48). 200 μL / well was added, the mixture was incubated at room temperature for 1 hour, and the well was blocked. Next, the plate was washed three times with 300 μl of PBS-T using a plate washer. Each sample was placed in 1% BSA-containing PBS-T with 2 of 8-fold diluted those plates end well, to prepare a dilution series for 2-fold serial dilutions to the other end, was incubated for 2 hours at room temperature . The blank was PBS-T containing 1% BSA. After the incubation, the plate was washed four times with 300 μL of PBS-T using a plate washer. Subsequently, a mixture of Goat anti-Human IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3 (Southern Biotech), which was 4000-fold diluted with PBS-T containing 1% BSA, was added at 100 μl / well and added at room temperature. Incubated for 1.5 hours. Thereafter, the plate was washed four times with 300 μl of PBS-T using a plate washer. A mixture of TMB Substrate and TMB Solution (seracare, 5120-0050) in an equal volume was added at 100 μl / well, and after 30 minutes of color development reaction, 50 μl of 2N H 2 SO 4 (nacalai tesque, 32520-55) was added. The reaction was stopped. The value of OD450 was measured with a plate reader, and the value of log2titer was calculated. The cut-off value was the average value of the blank well + 0.1.
結果
１．結核菌ワクチン
（１）Ag85B抗原
　アジュバント活性を発揮することが知られているCpGK3 10μgと、STINGリガンドの１種であるcGAMP 1μgの混合物を添加した場合と比較して、cyclic-di-GMP 10 μgで同程度の抗原特異的なTh1細胞性免疫の誘導が観察された（図１）。STINGリガンド単体（cAMP、GMP、およびcGAMP）での比較においては、cyclic-di-AMP（cAMP）が比較的有効と思われた。 Result 1. Mycobacterium tuberculosis vaccine (1) Ag85B antigen 10 μg of cyclic-di-GMP compared to the case where a mixture of 10 μg of CpGK3, which is known to exhibit adjuvant activity, and 1 μg of cGAMP, which is a kind of STING ligand, is added. In a similar degree, induction of antigen-specific Th1 cell-mediated immunity was observed (FIG. 1). In comparison with the STING ligand alone (cAMP, GMP, and cGAMP), cyclic-di-AMP (cAMP) appeared to be relatively effective.
　次に、STINGリガンドをアジュバントとして使用した場合のTh1細胞およびTh17細胞の誘導効果について検討した。STINGリガンドとしてCyclic-di-GMPを用いた。Cyclic-di-GMPを含まないワクチン抗原を投与したマウスでは、抗原特異的Th1細胞およびTh17細胞はほとんど誘導されなかった（図２および図３「cCHP-Ag85B」）。また、抗原提示細胞を抗原で刺激しなかった場合にもT細胞はほとんど誘導されなかった。一方、肺および脾臓においては、cCHP-Ag85B+cyclic-di-GMPの経鼻投与により顕著に抗原特異的なTh1およびTh17が誘導され、全身性免疫応答および粘膜免疫応答の両者が効率よく誘導されてくることがわかった（図２および図３）。 Next, the effect of inducing Th1 cells and Th17 cells when the STING ligand was used as an adjuvant was examined. Cyclic-di-GMP was used as a STING ligand. Mice to which a vaccine antigen containing no Cyclic-di-GMP was administered hardly induced antigen-specific Th1 cells and Th17 cells (FIGS. 2 and 3 "cCHP-Ag85B"). T cells were hardly induced even when the antigen-presenting cells were not stimulated with the antigen. On the other hand, nasal administration of cCHP-Ag85B + cyclic-di-GMP induced marked antigen-specific Th1 and Th17 in the lung and spleen, and efficiently induced both systemic and mucosal immune responses. (FIGS. 2 and 3).
　STINGリガンドをアジュバントとして使用した本発明のナノゲル経鼻ワクチンをマウスに投与した場合の生存率と結核菌の増殖に及ぼす影響について、BCGワクチンをポジティブコントロールとして調べた。感染後から12週までの間で死亡例が数例観察されたことから生存率を算出したところ、未免疫マウス（ネガティブコントロール）56％、BCGワクチン群（ポジティブコントロール）67％に比して、ナノゲル群（cCHP-Ag85B+cyclic-di-GMP投与）が89％と感染に抵抗性を示した（図４Ａ）。また、脾臓における結核菌数は、未免疫マウスと比較して、BCGおよびナノゲルワクチン群で同等に有意に菌の増殖が抑制されており、肺でも同様の傾向が観察された（図４Ｂ）。 B The BCG vaccine was examined as a positive control for the effect on the survival rate and growth of M. tuberculosis when the intranasal nanogel vaccine of the present invention using the STING ligand as an adjuvant was administered to mice. The survival rate was calculated from the fact that several deaths were observed from the 12th week after infection to 56% of non-immunized mice (negative control) and 67% of BCG vaccine group (positive control). The nanogel group (cCHP-Ag85B + cyclic-di-GMP administration) showed 89% resistance to infection (FIG. 4A). In addition, the number of M. tuberculosis bacteria in the spleen was significantly significantly suppressed in the BCG and nanogel vaccine groups as compared with the non-immunized mice, and a similar tendency was observed in the lungs (FIG. 4B).
（２）ESAT6- Rv2660c-Rv0288キメラ抗原
　cCHP-キメラ＋cyclic-di-AMPの経鼻的な投与により、脾臓および子宮頸部において抗原特異的Th1細胞が誘導されてくることがわかった（図５）。また、cCHP-キメラのみの投与では抗原特異的Th1はいずれの臓器でも誘導されてこなかったことから、このTh1にはcyclic-di-AMPが必須と考えられた。 (2) Nasal administration of ESAT6-Rv2660c-Rv0288 chimeric antigen cCHP-chimera + cyclic-di-AMP was found to induce antigen-specific Th1 cells in the spleen and cervix (FIG. 5). . In addition, administration of cCHP-chimera alone did not induce antigen-specific Th1 in any of the organs. Thus, it was considered that cyclic-di-AMP was essential for Th1.
２．HPVワクチン
（１）cyclic-di-AMPをアジュバントとした場合
 HPVの変異型E7タンパク質を抗原として、本発明のナノゲル経鼻ワクチンを作製し、このワクチンのT細胞等の誘導効果について検討した。
　cCHP-変異型E7＋cyclic-di-AMPの経鼻的な投与により、脾臓および子宮頸部において抗原特異的CTLが誘導されてくることがわかった（図６）。また、cCHP-変異型E7＋cyclic-di-AMPの経鼻的な投与により、脾臓および子宮頸部において抗原特異的Th1細胞が誘導されてくることがわかった（図７）。 2. HPV vaccine (1) In the case of using cyclic-di-AMP as an adjuvant The nanogel nasal vaccine of the present invention was prepared using HPV mutant E7 protein as an antigen, and the effect of inducing the vaccine on T cells and the like was examined.
Nasal administration of cCHP-mutant E7 + cyclic-di-AMP was found to induce antigen-specific CTL in the spleen and cervix (FIG. 6). In addition, it was found that intranasal administration of cCHP-mutant E7 + cyclic-di-AMP induced antigen-specific Th1 cells in the spleen and cervix (FIG. 7).
（２）STINGリガンド3種（cyclic-di-GMP, cyclic-di-AMP, cGAMP）をアジュバントとした場合
　cCHP-変異型E7タンパク質抗原と組み合わせた粘膜アジュバントにおいて、なかでも3種のSTINGリガンドと組み合わせた経鼻免疫において、それぞれ子宮頸部で抗原特異的Th1（図８右）およびCTL（図８左）が誘導されてくることがわかった。Th1誘導においてはSTINGリガンド間で大きな差は認められず、CTLではcyclic-di-AMPによる誘導が強く観察された。 (2) When three types of STING ligands (cyclic-di-GMP, cyclic-di-AMP, cGAMP) are used as adjuvants In mucosal adjuvants combined with cCHP-mutant E7 protein antigen, three types of STING ligands are combined In nasal immunization, it was found that antigen-specific Th1 (right in FIG. 8) and CTL (left in FIG. 8) were respectively induced in the cervix. In Th1 induction, no significant difference was observed between STING ligands. In CTL, induction by cyclic-di-AMP was strongly observed.
３．RSVワクチン
　SHペプチドに対する抗体およびキャリアタンパク質であるPspAに対する抗体いずれも経鼻免疫の回数に伴い、経時的に上昇することがわかった（図９）。また、SHペプチド特異的な免疫応答では、cyclic-di-AMPを加えた群においてよりIgG誘導が顕著ではあるが（図９左）、cyclic-di-AMPなしのcCHP-PspA-SH3投与のみの群においても、免疫の回数依存的に特異的抗体の誘導が観察された。
　IgGサブクラスでは、抗SHペプチドおよび抗PspA抗体のいずれにおいても、cyclic-di-AMPアジュバントなしでIgG1が優位に、アジュバントありでIgG1およびIgG2bが誘導されてきた（図１０）。 3. Both antibodies against the RSV vaccine SH peptide and antibodies against the carrier protein PspA were found to increase over time with the number of intranasal immunizations (FIG. 9). In the SH peptide-specific immune response, IgG induction was more remarkable in the group to which cyclic-di-AMP was added (FIG. 9 left), but only cCHP-PspA-SH3 administration without cyclic-di-AMP was performed. In the group as well, induction of specific antibodies was observed depending on the number of times of immunization.
In the IgG subclass, in both the anti-SH peptide and the anti-PspA antibody, IgG1 was predominant without cyclic-di-AMP adjuvant, and IgG1 and IgG2b were induced with adjuvant (FIG. 10).
　以上のように、ナノゲルワクチンにアジュバント（本実施例ではSTINGリガンド）を封入して投与すると、Th1細胞やCLTなど細胞性免疫に特徴的なT細胞が誘導された。さらに、全身性免疫のみならず上気道下気道粘膜組織に加え、生殖器粘膜組織における粘膜免疫を誘導することも明らかとなった。 As described above, when an adjuvant (STING ligand in this example) was encapsulated and administered in the nanogel vaccine, T cells characteristic of cellular immunity, such as Th1 cells and CLT, were induced. Furthermore, it was also found that it induces mucosal immunity not only in systemic immunity but also in genital mucosal tissues in addition to upper respiratory and lower respiratory tract mucosal tissues.
　本発明のナノゲル経鼻ワクチンは、細胞性免疫を誘導することができるため免疫細胞療法などの医学分野における利用が期待される。 The nanogel nasal vaccine of the present invention can induce cell-mediated immunity and is expected to be used in medical fields such as immunocell therapy.

Claims (11)

  1. 　ナノゲル、ワクチン抗原およびアジュバントの複合体を含むワクチン製剤。 ワ ク チ ン Vaccine preparation containing a complex of nanogel, vaccine antigen and adjuvant.
  2. 　前記アジュバントが１または複数のSTINGリガンドを含むことを特徴とする請求項１に記載のワクチン製剤。 ワ ク チ ン The vaccine formulation according to claim 1, wherein the adjuvant contains one or more STING ligands.
  3. 　前記STINGリガンドの少なくとも１つが、環状ジヌクレオチドであることを特徴とする請求項２に記載のワクチン製剤。 ワ ク チ ン The vaccine formulation according to claim 2, wherein at least one of the STING ligands is a cyclic dinucleotide.
  4. 　前記環状ジヌクレオチドが、cGAMP、cyclic-di AMP、cyclic-di GMP、cyclic-di CMP、cyclic-di UMPまたはcyclic-di IMPのいずれかであることを特徴とする請求項３に記載のワクチン製剤。 The vaccine preparation according to claim 3, wherein the cyclic dinucleotide is cGAMP, cyclic-dicyclicAMP, cyclic-di GMP, cyclic-di CMP, cyclic-di UMP or cyclic-di IMP. .
  5. 　前記ワクチン抗原が結核菌由来の抗原であることを特徴とする請求項１ないし４のいずれかに記載のワクチン製剤。 ワ ク チ ン The vaccine preparation according to any one of claims 1 to 4, wherein the vaccine antigen is an antigen derived from Mycobacterium tuberculosis.
  6. 　前記結核菌由来の抗原が、少なくともAg85B遺伝子産物、Rv2608遺伝子産物、Rv3619遺伝子産物、Rv3620遺伝子産物、Rv1813遺伝子産物、MTB32A遺伝子産物、MTB39A遺伝子産物および／またはMVA85A遺伝子産物の全体もしくはその一部を含むことを特徴とする請求項５に記載のワクチン製剤。 The antigen derived from Mycobacterium tuberculosis comprises at least the whole or a part of the Ag85B gene product, the Rv2608 gene product, the Rv3619 gene product, the Rv3620 gene product, the Rv1813 gene product, the MTB32A gene product, the MTB39A gene product, and / or the MVA85A gene product. The vaccine preparation according to claim 5, characterized in that:
  7. 　前記結核菌由来の抗原が、Rv3875遺伝子産物、Rv0266遺伝子産物およびRv0288遺伝子産物からなるキメラタンパク質であることを特徴とする請求項５に記載のワクチン製剤。 (6) The vaccine preparation according to (5), wherein the M. tuberculosis-derived antigen is a chimeric protein consisting of Rv3875 gene product, Rv0266 gene product, and Rv0288 gene product.
  8. 　前記ワクチン抗原がHPV（human papillomavirus）由来の抗原であることを特徴とする請求項１ないし４のいずれかに記載のワクチン製剤。 The vaccine preparation according to any one of claims 1 to 4, wherein the vaccine antigen is an antigen derived from HPV (human papillomavirus).
  9. 　前記HPV由来の抗原が少なくともE6遺伝子産物および／またはE7遺伝子産物の全体もしくはその一部を含むことを特徴とする請求項８に記載のワクチン製剤。 The vaccine preparation according to claim 8, wherein the HPV-derived antigen contains at least the whole or a part of the E6 gene product and / or the E7 gene product.
  10. 　前記ワクチン抗原がRSV（respiratory syncytial virus）由来の抗原であることを特徴とする請求項１ないし４のいずれかに記載のワクチン製剤。 The vaccine preparation according to any one of claims 1 to 4, wherein the vaccine antigen is an antigen derived from RSV (respiratory syncytial virus).
  11. 　前記RSV由来の抗原が少なくともSHペプチドの全体もしくはその一部を含むことを特徴とする請求項１０に記載のワクチン製剤。 11. The vaccine preparation according to claim 10, wherein the RSV-derived antigen contains at least the whole or a part of the SH peptide.
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